Search

Human apolipoprotein A-II (apo A-II)consists of throe potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of ibis apolipoprotein. The conformation and lipidbinding properties of these peptides either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and arc compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides to define the minimal peptide length required for stable complex formation The properties of the apo-A-II-(l3-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(l3-30)-(33-48)peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix 13 48 was mi active either. The apo-A-II-(37-77)-peptide and turn-he:l ix segment spanning residues - the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-773)-peptide, which consisted of the C-terminal helix, a β-turn and wart of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo A-II-(-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however; less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association. [ABSTRACT FROM AUTHOR]

Lipid bodies are cellular compartments containing triacylglycerols. They are encompassed by a phospholipid monolayer and decorated with characteristic proteins. In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination. In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase. We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogens bromide. A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forms during germination. This allowed a clear distinction of this enzyme from other lipoxygenase isoforms. Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day I of germination. From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on nothern blots. The very early expression of lipid body lipoxygenase was corroborated by this approach. Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically. In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro-translated lipoxygenase and the lipid body form. [ABSTRACT FROM AUTHOR]

Amphipathic helical repeals arc considered as the structural units of numerous apolipoproteins and have been described as being responsible for the interaction of apolipoproteins with phospholipids in high-density lipoproteins (HDL). Furthermore. apolipoproteins. and especially apolipoprotein Al (apoAl), are involved in various biological functions of these circulating particles in plasma. Studies with synthetic peptides corresponding to domains of the apoAl sequence have however shown that short 39-residue fragments do not interact strongly enough with phospholipids to generate particles that correctly mimic the physicochemical properties of HDL reconstituted with native apoAl [Vanloo, B., Demoor, L., Boutil- Ion. C.. Lins, L.. Baert, J,, Fruchart, J. C.. Tartar. A. & Rosseneu, M. (1995) Association of synthetic peptide fragments of human apolipoprotein A-I with phospholipids. i. Lipid Res. 36. 1686-1696.1. Here we show that synthetic branched multirneric peptides. often used as carriers for the design of synthetic vaccines (multiple-antigen peptides). can be used to mimic the physicochemical properties of apoAl in HDL. This type of molecule is obtained by using a small core matrix of Lys residues bearing radially branched synthetic peptides as dendritic arms. We compared the lipid-binding capacities and the structural properties of a linear peptide corresponding to residues 145--183 of apoAl [apoAl-(145-183)-peptide) with those of two multimeric peptides consisting respectively of three [trimeric apoAl-(145-183)j and four copies [tetrameric apoAl-( 145- 183)] of the selected sequence. branched on a covalent core matrix. This paper provides evidence for the increased abilities of the multimeric peptides to associate with phospholipids compared with the short linear peptides. Moreover, the trimeric apoAl-(145 - 183) peptide was most efficient in mimicking the physicochemical and structural properties of native apoAl in reconstituted HDL. As tools adequate to unravel the structure/function relationship of separate apolipoprotein domains are still missing. these multirneric peptides might constitute an alternative approach to linear peptides which are poor mimetics and to protein mutants which are difficult to produce and only provide information about the total sequence. [ABSTRACT FROM AUTHOR]

Recently it was demonstrated that the metabolism of both glycoproteins and sphingo(glyco)lipids is dependent upon the state of enterocytic differentiation of HT-29 cells. Furthermore, it was shown that undifferentiated HT-29 cells display an important autophagic sequestration, controlled by a heterotrimeric G1, protein. In order to correlate the metabolism of sphingo(glyco)lipids with the extent of autophagic sequestration, we have incubated undifferentiated and differentiated HT-29 cells with tritium-labelled GM1 ganglioside and sphingosine in the absence and presence of pertussis toxin (an inhibitor of autophagic sequestration) or asparagine (an inhibitor of autophagic vacuole maturation). In addition, undifferentiated HT-29 cells transfected with a cDNA encoding the GU1 protein (cells expressing an amplified autophagic pathway) were labelled with both GMI and sphingosine. The result show that the catabolism of sphingo(glyco)lipids is dramatically enhanced in parallel with the increase of the autophagic pathway while at the same time their biosynthesis is reduced. The inhibition of autophagy in both undifferentiated cells and α1-overexpressing cells restores sphingo(glyco)lipid metabolism, as normally expressed in differentiated cells, as well as in other mammalian cell types. We conclude that autophagy plays an important role in governing the metabolic fate of sphingo(glyco)lipids in HT-29 cells. Since autophagy regulates the N-linked glycoprotein metabolism in this cell line, our results corroborate the idea that glycolipid and glycoprotein metabolisms are controlled by similar mechanisms. [ABSTRACT FROM AUTHOR]

A cytosolic 21-kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. It was encountered in several tissues and species; however, its accurate function remained partially unknown. In order to obtain information from its structural features, we built a molecular model which revealed it to possess a nucleotide-binding site. In the present research, we describe the affinity of the bovine brain 21-kDa protein for nucleotides, and its association with cytosolic proteins, small GTP-binding proteins and lipid droplets. Our results suggest that, through its association with small GTP-binding proteins, the 21-kDa protein is implicated in signal mechanisms during cell growth and maturation. [ABSTRACT FROM AUTHOR]

Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacterium halobrium, was used and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2GroPCho) and distearoyl- (Ste2GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-1,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsin/phosphatidylcholine molar ratios ≈ 1:50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length dv ≈ 3.0–3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau2GroPCho (ΔT ≈ 40 K, mean hydrophobic thickness &dmacr; ≈ 2.4 nm), and to a lesser extent of Myr2GroPCho (ΔT ≈ 23 K, &dmacr; ≈ 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam2GroPCho (ΔT ≈ 0 K, &dmacr; ≈ 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (ΔT ≈ −13 K, &dmacr; ≈ 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsin/phosphatidylcholine molar ratios < 1:500). In this region and for Lau2GroPCho, both the spectroscopic data and circular-dichroism spectra indicated that the protein was in the monomeric form. Phase diagrams, in temperature versus bacteriorhodopsin concentration, were constructed for Lau2GroPCho and Ste2GroPCho. On account of microscopic theoretical models and of the relative values of dp and d, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2GroPCho but in the liquid disordered state in Ste2GroPCho. The phase diagram of Lau2GroPCho was also analyzed using another theoretical approach based upon elastic models within the Landaude Gennes theory. This allowed for the estimation of the coherence length ζ which characterizes the distance over which the hydrophobic thickness of the lipid bilayer is perturbed by the protein. A value of 1.2 nm was found, agreeing relatively well with theoretical predictions. [ABSTRACT FROM AUTHOR]

Colicins A and N are pore-forming bacterial toxins that kill Escherichia coli cells. Their mode of action involves three steps; binding to specific receptors located in the outer membrane, translocation through this membrane and the periplasm, and channel formation in the inner membrane. In-vitro studies have shown that negatively charged phospholipids are an absolute requirement for the channel formation of colicin A. Using HDL11 strain, in which the phosphatidylglycerol (PtdGro) content was altered by varying the synthesis of the PtdGro-phosphate synthase, the effect of envelope PtdGro content on the activity of colicin A was studied in vivo. The formation by colicin A of a voltage-gated channel in the cytoplasmic membrane results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed by the toxin to cross the cell envelope. This lag time is higher when the cells have a reduced PtdGro level, suggesting that the receptor/translocation machinery of colicin A (OmpF, BtuB and Tol QRAB proteins) is altered in the absence of PtdGro. The rate of potassium efflux is also greatly reduced when the PtdGro content is decreased, suggesting that a certain level of PtdGro is indeed required for proper insertion of the colicin-A channel. In contrast, the activity of colicin N does not show any PtdGro dependence. The difference between the behavior of colicin A and that of colicin N is discussed. [ABSTRACT FROM AUTHOR]

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x l05 M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 µM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine < oleic acid < phosphatidie acid < phosphatidylglycerol < phosphatidylmethanol < phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from I nM at 2.5 µM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 µM phospholipid. The Vmax increased from 20 mol factor Va inactivated · min-1 · mol APC-1 at 2.5 µM phospholipid to 62 mol factor Va inactivated · min-1 · mol APC-1 at 10 µM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S. [ABSTRACT FROM AUTHOR]

Conformational properties and interactions with lipid membranes were studied for the chemically synthesized peptides PC(1–37) and PC(1–43), corresponding to the N-terminal 37 and 43 residues, respectively, of the transit peptide of the precursor to plastocyanin of Silence pratensis. PC(1–43) covers the entire chloroplast targeting domain of the transit peptide. CD spectra of PC(1–37) and PC(1–43) showed that both peptides have little ordered structure in aqueous solutions but form partially helical conformations in the presence of detergent micelles or in methanol. Vesicle disruption and direct-binding experiments revealed, however, that neither PC(1–37) nor PC(1–43) had a high affinity for lipid membranes. Since in the intact plastocyanin transit peptide the chloroplast-targeting domain is followed by a hydrophobic thylakoid-transfer domain, the plastocyanin precursor may well be transported to the chloroplast surface first with the aid of the thylakoid-transfer domain. The chloroplast-targeting domain may then form a helical structure in the lipid environments, and a chloroplast-specific motif displayed on the helical structure may be recognized by a receptor protein located at the chloroplast envelope membranes. [ABSTRACT FROM AUTHOR]

Glycophorin has been reconstituted into egg phosphatidylethanolamine (egg-PtdEtn) membranes. Stable vesicles were obtained at a molar ratio of 8 × 10-4 of inserted protein/lipid. This macroscopic change from lipid aggregates to lipid vesicles was followed by density gradient centrifugation. Vesicles formed in the presence of protein enclose the dye calcin and are stable with time and temperature. Membrane aggregation does not occur, as was demonstrated by energy-transfer experiments. The phase transition from the fluid lamellar Lα phase to the inverted hexagonal HII phase observed at 29° C in pure egg-PtdEtn membranes is suppressed and finally disappears in the presence of glycophorin. The transition enthalpy decreases linearly from ΔH = 4 kJ/mol in pure lipids to zero at a protein/lipid molar ratio of 1:1000. Ca2+ ions and wheat germ lectin act synergistically on the phase behavior of vesicles containing glycophorin and phosphatidylethanolamine. Differential scanning calorimetry scans show that the lamellar-to-hexagonal phase transition is reinduced. The membranes aggregate and exchange lipid as could be demonstrated by energy transfer experiments. The dye calcin is released but only if the temperature exceeds the lamellar-to-hexagonal phase transition temperature of the pure egg-PtdEtn. [ABSTRACT FROM AUTHOR]

Staphylococcal δ-toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self-association process is observed. Analytical ultracentrifuge and quasi-elastic light-scattering experiments suggest an isodesmic aggregation m the high concentration domain above 2 μM up to very large asymmetrical species. Decreasing concentrations below 2 μM of δ-toxin and the analogue allows dissociation, probably into monomers. The self-associated species are essentially α-helical (70%) with buried and highly immobilized Trp either at position 15 for natural δ-toxin or 16 for the analogue. At the lowest concentration studied, the α-helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11–26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods. When bound to lipids, whatever their initial structure in solution, 26-residue long peptides mainly adopt an α-helix conformation (80%) while fragment 11–26 exhibits about 50% α-helix. The lipid-peptide interactions were quantitatively analysed. For fragment 11–26, a single-step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26-residue-long analogue, multiple-step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11–26 severely decreases from micelles to fluid and gel-state bilayers. The partition coefficient of the δ-toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that δ-toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing. [ABSTRACT FROM AUTHOR]

The protein translocation system of Escherichia coil was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of lgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined, IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1- ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation. [ABSTRACT FROM AUTHOR]

Bovine heart bc1 complex was reversibly inactivated by a new simple and effective chromatographic delipidation method. Upon phospholipid replenishment, catalytic activity increased from values near zero to values 2-6-times higher than those of the original preparation. Compared to original preparations maximally activated by additional phospholipid, the degree of reactivation was up to 100%. By this delipidation method, the 6.4-kDa protein subunit was removed with the phospholipid. The loss of this protein neither diminished electron transport activity nor abolished proton translocation. Two requirements were necessary to obtain quantitative data: (a) only bc1 complexes, homogeneously dissolved before and after relipidation had to be used and (b) the phospholipid bound to the complex had to be determined. The correlation of catalytic activity to bound phospholipid was studied in the range of low phospholipid/ protein ratios, which had previously been insufficiently resolved. Catalytic activity increased linearly with added phospholipid up to a molar ratio of 80-100 lipid molecules/dimeric complex. This corresponds to the number of phospholipid molecules that complete a single bilayer annulus. The activating effect of phospholipid is not merely due to a hydrophobic phase effect, since it strongly depends on the nature of the polar head group of the added phospholipid. Of the three major phospholipids bound to the bc1 complex, only phosphatidylethanolamine and phosphatidylcholine activated when added as sole phospholipid. Tightly bound diphosphatidylgtycerol was needed for preservation of the native complex structure. [ABSTRACT FROM AUTHOR]

We have sequenced the nucleotides of the gene POX18 that encodes PXP-18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13 805 Da), was identical to that of the nonspecific lipid-transfer protein (sterol carrier protein 2) from rat liver. PXP-18. purified to near homogeneity from isolated peroxisomes, had an amino-terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid-transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP-18 lacked tile cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP-18 has a role in the β-oxidation of long-chain fatty acids. PXP-18 modulated acylcoenzyme A oxidase activity at low pH. [ABSTRACT FROM AUTHOR]

The interaction of the non-ionic surfactant Triton X-100 with Halobacteriurn purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a halftime of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from alttrans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacterioopsin Schiff's base (bleaching). [ABSTRACT FROM AUTHOR]

Addition of EDTA to mitochondria incubated aerobically in a phosphate-supplemented medium containing Na+ ions results in activation of cation uptake which is accompanied by membrane depolarization and stimulation of respiration. The same results are obtained in media containing Li+ but not K+, indicating that this pathway for cation transport is selective. The activation of Na+ transport is not accompanied by changes of matrix Mg+. indicating that cation transport is controlled by surface-bound rather than intramitochondrial Mg2+. Na+ transport in respiring mitochondria is competitively inhibited by Mg2+ with a K1 in the nanomolar range. A Na+ current can also be induced by a K+ diffusion potential in the absence of respiration. The K+-diffusion-driven Na+ current has the same magnitude in the absence or presence of inorganic phosphate, suggesting that Na+ transport is mediated by Na+ uniport rather than by electrogenic nNa+/H+ antiport with n > 1. Analysis of the flow/force relationship indicates that the putative Na+ uniporter has a conductance of about 0.2 nmol Na+ × mg protein-1 × min-1 × mV-1, and that it is active only when the membrane potential exceeds about 150 mV. [ABSTRACT FROM AUTHOR]

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and ²H20 media, the following percentage distribution of secondary structure patterns has been obtained: 50% α-helix, 28–30% β-structure; 13–15% β-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In acordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide l maximum and the appearance of two new peaks in ²H20, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40–50°C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However. no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of α-helix, probably corresponding to t he segments spanning the membrane, but the conformation of the polar domains appears to differ. [ABSTRACT FROM AUTHOR]

The acyl-carrier protein (ACP) in Neurospora crassa mitochondria [Brody, S. & Mikolajczyk, S. (1988) Eur. J. Biochem. 173, 353–359] mediated a cerulenin-sensitive, de novo fatty acid synthesis independent of the fatty acid synthetase complex present in the cytoplasm. Incubation of mitochondria with 12-14C]malonate labeled only the ACP as indicated by autoradiography after SDS/PAGE. Under these in vitro conditions ATP was required for the initial acyl-ACP formation, but further elongation required either magnesium or the direct addition of NADPH. Labeled hexanoic (6:0) and caprylic (8:0) acids were detected as intermediates in the pathway, as well as hydroxymyristic acid. All of the intermediates, and the eventual product of the reaction, myristic acid (14:0), were released from the ACP by alkaline treatment. Pulse-chase experiments demonstrated the incorporation on to, and release of label from, the ACP. In vivo labeling of ACP with [2-14C]malonate was also detected and the label was in the form of hydroxymyristic acid. This newly discovered pathway is discussed from the standpoint of its possible role in providing acyl chains for mitochondrial lipids. [ABSTRACT FROM AUTHOR]

Cholesteryl-ester-fich very-low-density lipoproteins (β-VLDL) are considered to be atherogenic because in vitro they can provoke cholesterol accumulation in macrophages. The greatest population ofmacrophages resides inside the liver and in the present study the rat β-VLDL uptake by the various rat liver cell types is determined in vivo and compared to the uptake of rat VLDL. β-VLDL isolated from cholesterol-fed rats was iodinated and injected into the rat. After 10 min of circulation, 45% of the injected β-VLDL was found in the liver. A low-temperature cell-isolation procedure shows that rat liver parenchymal cells form the major site for β-VLDL uptake (96%) and, consequently, rat liver macrophages (nonparenchymal liver cells) do not perform a quantitatively significant role in the uptake of these lipoproteins. In vitro competition studies indicate that apolipoprotein (apo) E is the site recognised by liver parechymal cells and even a 600-fold excess of apo-E-free human LDL was an ineffective competitor. Furthermore it can be demonstrated that induction of apo-B,E receptors on liver parenchymal cells by estrogen treatment does not result in a significant increased uptake of β-VLDL. These data show that recognition of β-VLDL is presumably exerted by the remnant receptor. Intracellular processing of both the apolipoproteins and phospholipids of β-VLDL was followed by subcellular distribution studies. It appears that, within 45 min, 75% of the apolipoproteins are degraded and subsequently released from the liver. In contrast the phospholipids remain associated with the liver for a prolonged time and a specific transfer to the mitochondrial fraction is found. It can be concluded that liver parenchymal cells form in vivo the major site for β-VLDL uptake and it appears that recognition of β-VLDL is coupled to internalization and processing of both the apolipoproteins and phospholipids by a route which involves the lysosomes. [ABSTRACT FROM AUTHOR]

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges. [ABSTRACT FROM AUTHOR]

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc, (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide. perturbing the phospholipids close to it, but allowing rapid (> 104 s-1) exchange with all the lipid molecules in the system. [ABSTRACT FROM AUTHOR]

Detergent-solubilized preparations of the β-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by α-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[γ-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex. [ABSTRACT FROM AUTHOR]

Replacement of the normal culture liquid to a nitrate-free medium resulted in an immediate drop in the ratio of protein to lipid in isolated cell envelopes of Anacystis nidulans cells. The relative fluidity of the envelope membranes or liposomes, made from the extracted lipids of the envelope, was estimated by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. A thermotrophic phase transition of lipids within the cytoplasmic membrane of intact cells was also revealed by detecting the temperature-dependent absorption changes in the proportion of zeaxanthin at 390 nm. It became evident that a decrease in the proportion of protein to lipid within the cell envelope was accompanied neither by changes in the microviscosity level, nor by shifting of characteristic temperatures of the liquid-crystalline-to-gel transition of lipids. In parallel with nitrate starvation, however, the proportion of saturated fatty acids of the envelope lipids increased markedly. Accumulation of saturaged, longer-chain (C18) fatty acids at the cost of C16 counterparts upon nitrate deprivation occured in all of the complex lipids. In accordance with these findings, a pronounced decrease in the fluidity was demonstrated for the liposomes prepared from the envelope polar lipids of nitrate-starved cells compared with corresponding control, throughout the temperature range (45-5°C) studied. We propose that the fluidizing effect due to a fall in the ratio of protein to lipid was compensated by a rapidly triggering regulatory process which enables the preservation of the fluidity characteristics at an optimal level within the cell envelope of A. nidulans. [ABSTRACT FROM AUTHOR]

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5° and 58°C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41°, 46°, 44° and 47°C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4′-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52°C for E. coil lipid A). The analysis of >CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22°, 30°, 27° and 25.5°C (first transition) and at about 34°, 42°, 38.5° and 40.5°C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: 1. Up to about 30°C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of >CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions. 2. At elevated temperatures no straightforward interpretation of the data was possible: while the X-ray data did not indicate any drastic changes in the bilayer arrangement at the molecular level even after completion of the acyl chain melting process, the fluorometric and calorimetric data suggested the existence of more complex supermolecular arrangements at temperatures above the main transition. [ABSTRACT FROM AUTHOR]

Outer membrane pore protein OmpC was identified as the receptor for the temperate Escherichia coli phage HK253hrk. The part of OmpC protein recognized by the phage was identified by using hybrid proteins in which parts of OmpC protein are replaced by the corresponding parts of the related PhoE protein. In contrast to other OmpC-specific phages, HK253hrk recognizes a part of OmpC within the C-terminal 50 amino acids of the protein. E. coli strains lysogenic for HK253hrk produce reduced amounts of OmpC protein, and produce a new pore protein instead. Expression of this new protein was temperature-dependent, i.e. low at 30 °C. The functioning of this new pore protein was characterized both in vivo by studying the uptake of β-lactam antibodies and in vitro after reconstitution of the protein in black lipid films. Its effective pore size was larger than that of the OmpF pores of E. coli B. The new porin appears to be cation-selective. A comparison with the selectivity of the known OmpC and OmpF pores of E. coli showed that the new pore has a higher selectivity than OmpF but is less selective than OmpC. The new pore protein appears to function in E. coli K12 lysogens as the receptor for the phages HK187, HK189 and HK332. [ABSTRACT FROM AUTHOR]

Lipid bodies from fat-mobilizing cotyledons of cucumber and other Cucurbitaceae were investigated Proteins and glycoproteins were analyzed by electrophoresis and then used to characterize the lipid body membrane at different stages of cell development. Contaminations by other membranes or organelles were ruled out by comparing the main constituents from the endoplasmic reticulum, cytosol, glyoxysomes and protein bodies with the pattern of the lipid body membrane, considering both the prevalent peptides and the dominating glycoproteins. Among the proteins of lipid body membranes in ripening and germinating cotyledons, a 90-kDa peptide was found as unique marker of lipid bodies at the stage of fat mobilization. The 90-kDa protein was purified, and antibodies against it were raised in rabbits. By means of immunoprecipitation and electrophoretic analysis it was demonstrated that the synthesis of the 90-kDa form located in lipid bodies shows a transient increase and subsequent decline, with maximal values being observed at day 3 of germination. At this stage, the rate of de novo synthesis was compared considering lipid body proteins and other organellar proteins. The 90-kDa protein appeared as the lipid body constituent that is synthesized and assembled in the organelle by far at the highest rates. [ABSTRACT FROM AUTHOR]

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high-molecular-mass product (120-140 kDa) was observed tinder mild conditions of solubilization, a folded monomeric firm of 35 kDa was seen when treated at high SDS concentrations and finally, a single hand around 45 kDa occurred when the sample was heated to 96°C [Bolla, J. M., Lorent, E., Zalewski, M. & Pages, J. M. (1995) J. Bacteriol. 177, 4266-4271]. The high-molecular-mass product was reconstituted into two-dimensional crystals in the presence of phospholipids and Mg2+. The C. jejuni porin required different conditions for successful reconstitution into two-dimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a = 8.9 nm, b = 14.9 nm, an oblique Lattice with a = 8.9 nm, b = 30.1 nm, γ = 98° and a trigonal lattice with a = 8.9 nm, b = 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain-filled indentations. [ABSTRACT FROM AUTHOR]

The interaction of coal proteins from coated vesicles with model lipid membranes was examined using small unilamellar vesicles of dipalmitoylglycerophosphocholine as model membranes. Changes in membrane permeability were measured by the leakage of entrapped fluorescent dye, carboxyfluorescein. Both clathrin and the 55000-Da protein were found to be active. Density gradient centrifugation showed the formation of art irreversible protein-lipid complex. Dynamic light-scattering measurements showed that this complex, is significantly Larger than the original vesicles, suggesting that fusion is induced. The effects of pH, urea, Tris and ionic strength were studied and the possible biological relevance of the results is discussed. [ABSTRACT FROM AUTHOR]

The lipophilic fluorescent probe diphenylhexatriene was used to probe the lipid order and dynamics in apolipoprotein C-I dimyristoylglycerophosphocholine (Myr2Gro-P-Cho) complexes. These complexes contain on the average 25 mol Myr2Gro-P-Cho/mol of apolipoprotein C-I, have a molecular weight around 200 000, and appear as discoidal, stacked particles by negative-stain electron microscopy. Steady-state fluorescence polarization of diphenylhexatriene as a function of temperature gives a broadened and shifted phase transition for Myr2Gro-P-Cho from the gel to liquid-crystalline state, with a mid-point around 27°C. Time-dependent fluorescence intensity and anisotropy measurements of the diphenylhexatriene probe at 15°C and 35°C give fluorescence decay curves which can best be fit by two exponential functions, in each case. The fluorescence lifetimes and their fractional amplitudes approach the corresponding parameters in Myr2Gro-P-Cho vesicles and suggest insignificant effects of the protein on the microenvironment and conformations of the probe. The rotational correlation times and their fractional anisotropies indicate similar local motions of the probe in complexes and in vesicles, but reveal a significant ordering effect of the protein at both temperatures. The overall complex rotation at 15°C has a correlation time of 136 ± 13 ns, consistent with the size (≈ 200 kDa) and shape (disc ≈ 5 x 15 nm) of the particle. [ABSTRACT FROM AUTHOR]

Unlike porcine pancreatic phospholipase A2, the enzyme from Naja melanoleuca does not display biphasic kinetic behaviour at substrate concentrations around the critical micelle concentration. This snake venom enzyme was further investigated by direct binding studies using n-tridecyiphosphocholine. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialysis and ultraviolet difference spectroscopy. It is concluded that, in the presence of submicellar concentrations of n-triclecylphosphochohne, a lipid-protein complex is formed consisting of about 4 protein and 36 lipid molecules. Ca2+ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. These lipid- protein aggregates are held responsible for the non-hyperbolic kinetic behaviour of the snake venom enzyme towards monomeric substrates. [ABSTRACT FROM AUTHOR]

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24°C, 28°C and 32°C with either pure dimyristoylglycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 0, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid- cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II. [ABSTRACT FROM AUTHOR]

The ‘functional’ state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [1-(5-dimethylaminonaphthalene)-sulfonamido]-n-hexanoic acid-β-N-trimethylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low-affinity state(s) for Dns-C6-Cho, similar to that found with the native membrane-bound receptor. This state can be slowly interconverted to a high-affinity state after rapid mixing with the agonist, and the non- competitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner. Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low-affinity state which can no longer be interconverted to a high-affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium chotate, a soluble (9 S) low-affinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane-bound receptor and in particular the slow interconversion to the high-affinity state and the effect of local anesthetics on this transition; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membrane-bound receptor and leads to the formation of vesicles which exhibit carbamylcholine-sensitive ion fluxes. A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent-soluble form. [ABSTRACT FROM AUTHOR]

The ‘reconstitution cycle’ is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a ‘reconstituted receptor’ fraction, under conditions which conserve part of the endogenous lipids, or ‘reconstituted vesicles’ in the presence of an excess of exogenous lipids. The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [3H]trimethyl-ammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor. Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles. With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of α-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle. [ABSTRACT FROM AUTHOR]

The lipid-A moiety of lipopolysaccharide induced freshly isolated bone marrow cells incubated with radioactive leucine to exhibit enhanced release of proteins other than immunoglobulins. Such stimulation by lipopolysaccharide was essentially not observed with cell suspensions from spleen, thymus, appendix, peritoneal exudate, whole blood, lymph node, liver, testis, buffy coat of blood and reticulocyte-enriched blood. The extracellular appearance of proteins was shown to be due to secretion by excluding other alternatives. Thus, the rate of cell death and/or leakage of cellular contents, as well as the rate of shedding of surface membrane protein, was unaffected measurably by lipopolysaccharide. Secretion of non-immunoglobulin proteins was selective as judged by the finding that the rate of immunoglobulin released by bone marrow cells during the usual four-hour pulse-label period was not stimulated by lipopolysaccharide. Enhancement of mitogenesis and enhancement of secretion of non-immunoglobulin protein by lipopolysaccharide appeared to occur at independent sites or even in different ceils because splenocytes which readily exhibited mitogenic response to lipopolysaccharide essentially did not exhibit stimulation of secretion of non-immunoglobulin protein. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide from pathogenic Yersinia pseudotuberculosis type III, strain 209, has been isolated and characterized. Lipopolysaccharide was shown to contain lipid A (30%), protein (4.4%), 2-keto-3-deoxyoctonate (7.8%) and a polysaccharide component (45%). which consisted of paratose, L-fucose, D-mannose, D-glucose, D-galactose (trace), L- and D-glycero-D-manno-heptose, 2-deoxy-2-amino-D-glucose and 2-deoxy-2-amino-D-galactose residues. Molecular-sieve chromatography on Sephadex G-50 afforded an O-specific polysaccharide, being hydrolysed to yield equimolar amounts of paratose, L-fucose, D-mannose and 2-deoxy-2-amino-D-galactose. The chemical repeating unit of the O-specific polysaccharide has been suggested on the basis of methylation studies and periodate oxidation data. [ABSTRACT FROM AUTHOR]

Evidence is presented for a non-covalent interaction between the spike glycoprotein and the nucleocapsid of Semliki Forest virus. When isolated viruses were treated with the non-ionic detergent β-D-octylglucoside at neutral pH and low ionic strength the lipid bilayer membrane could be removed leaving most of the spike glycoproteins attached to the nucleocapsids. The interaction between capsids and membrane protein was sensitive to elevated pH and ionic strength, but at least partially reversible at neutral pH and low ionic strength. The possible role of this interaction in the viral structure and in the mechanism of budding of the virus from the host cell is discussed. [ABSTRACT FROM AUTHOR]

Using conditions to avoid the utilization of labelled precursors by intracellular glycosyltransferases, experiments are described demonstrating that intact rat-spleen lymphocytes are capable of utilizing exogenous GDP-mannose and UDP-N-acetylglucosamine to synthesize dolichyl monophosphate mannose and dolichyl diphosphate oligosaccharides. Kinetic and chase experiments show that dolichyl diphosphate oligosaccharides are either utilized for the transfer of their carbohydrate moieties to protein acceptors or further degraded. Since glycosylation of proteins is limited in resting lymphocytes, the degradation pathway appears as a major event in the fate of the dolichyl diphosphate oligosaccharides synthesized in vitro. These dolichyl diphosphate oligosaccharides are degraded into phospho-oligosaccharides and oligosaccharides which are released in the medium. This enzymatic cleavage of the phosphodiester bond is inhibited by bacitracin. The phospho-oligosaccharides are susceptible to alkaline phosphatase giving neutral oligosaccharides and they are cleaved by endo-N-acetyl-β-D-glucosaminidase H leaving N-acetylglucosamine l-phosphate and neutral oligosaccharides. These data suggest that splitting of the phosphodiester bond of dolichyl diphosphate oligosaccharides, dephosphorylation and/or endo-N-acetyl-β-D-glucosaminidase hydrolysis of the phosphorylated oligosaccharides could represent the beginning of the catabolic pathway of dolichyl diphosphate oligosaccharides. [ABSTRACT FROM AUTHOR]

1. Dark incubation of retinoids (retinyl ester, retinol, retinal, retinaloxime) in suspensions of rod outer segment membranes leads to substantial isomerization (and partial degradation) in the case of retinals only. 2. All-trans, 13-cis and 9-cis-retinal all isomerize at the Δ13 double bond leading to an equilibrium with approximately 75% trans and 25% cis isomer at this bond (all-trans ↔ 13-cis and 9-cis↔9.13-dicis). 11-cis-Retinal isomerizes irreversibly to a mixture of all-trans and 13-cis-retinal. 3. The active compound appears to be phosphatidylethanolamine present in the membrane. The amino group and the phosphate, as well as the hydrophobic part of the phospholipid are essential. 4. At least three factors are important for the phosphatidylethanolamine-catalyzed isomerization as studied with the 13-cis isomer: the concentration of phosphatidylethanolamine, the concentration of Schiff base between retinal and phosphatidylethanolamine and the presence of lipid aggregates. 5. Based on these observations a mechanism is proposed, which satisfactorily explains the specificity of the isomerization pattern. 6. It is suggested that reisomerization of all-trans to 11-cis retinal in vivo takes place by fixation of all-trans retinal on an adequate surface (e.g. opsin) and a localized nucleophilic attack on the C-11 atom, followed by trapping of the isomerized chromophore by opsin. 7. It is further concluded that retinal does not occur in vivo as a free intermediate. Direct transfer from one protein to another (opsin, retinol dehydrogenase, retinal binding proteins) seems to take place. [ABSTRACT FROM AUTHOR]

The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles and rat liver microsomes, much higher concentrations of exchange protein were required in the case of intact red blood cells and microsomes. In human erythrocytes, 75% of the phosphatidylcholine was available for exchange within 2 h at 37 °C. No additional exchange was observed during the next 2 h, indicating slow, if any, transbilayer movement of the residual phosphatidylcholine. In rat erythrocytes 50–60% of the phosphatidylcholine was readily available for the exchange proteins. The residual phosphatidylcholine was exchanged at a much lower rate with a half time for equilibration of 7 h. These results confirm in an independent way the asymmetric distribution of phosphatidylcholine over the membrane of human and rat erythrocytes as well as the occurrence of a slow transbilayer movement of this lipid in rat erythrocytes. [ABSTRACT FROM AUTHOR]

Enzymatically inactive, delipidated Na,K-ATPase from dogfish rectal glands was titrated with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine. The process of relipidation has the following characteristic properties. Enzymatic activities reappear independently of each other: first the phosphatase, then the ATPase. The properties of the phosphatase regenerated depend on the ratio of lipid/protein used; the ATPase seems to be independent of this ratio. The simplest model that is consistent with the above results and with the shapes of the titration curves, has the following requirements. Firstly, the enzyme is composed of two subunits that, as far as lipid binding is concerned, are identical and independent of each other. Secondly, lipid adds onto the enzyme as preformed clumps of 25 molecules of phosphatidylcholine or 18 molecules of phosphatidylethanolamine. Thirdly, each subunit binds two clumps of lipid, and binding shows positive cooperativity. Fourthly, when either subunit becomes saturated with lipid, the enzyme exhibits one form of phosphatase. Fifthly, when both subunits are saturated with lipid, the enzyme exhibits a second form of phosphatase and ATPase. The data and their analysis according to this model lead to the suggestion that Na,K-ATPase is a functional dimer, the interaction between subunits being influenced by the Na+ and K+ concentrations in the medium: K+ favouring the functional independence of the subunits and Na+ favouring their functional interaction. [ABSTRACT FROM AUTHOR]

Crude membrane preparations from chick embryo cells catalyse the formation of dolichyl-di-N-acetylchitobiosyl diphosphate [Dol-PP-(GlcNAc)2] from uridine diphosphate jV-acetylglucosamine (UDP-GlcNAc). The formation of this glycolipid was stimulated by exogenous dolichyl phosphate and inhibited by tunicamycin. Adding GDP-mannose to the cell-free system containing Dol-PP- (GlcNAc)2 by preincubation led to the formation of a lipid-linked oligosaccharide, containing 8-9 sugar residues. The formation of lipid-linked oligosaccharides was inhibited by GDP-2-deoxy-Dglucose (GDP-dGlc): in this case Dol-PP-(GlcNAc)2-dGlc accumulated. Subsequent additions of mannosyl residues to this trisaccharide-lipid to form lipid-linked oligosaccharides were not possible. Concomitantly the glycosylation of proteins was blocked. Partially inhibitory conditions were obtained by adding both GDP-dGlc and GDP-Man with an excess of GDP-dGlc. Glycosylation of proteins was observed but the glycopeptides did not contain 2-deoxyglucosyl residues. Also in these cases 2-deoxyglucose-containing glycolipids accumulated. The main glycolipid formed under these conditions was Dol-PP-(GlcNAc)2-Man-dGlc. Lipid-linked oligosaccharides containing 2-deoxyglucose were formed under these conditions, although in small amounts, but were not transferred to protein. So the molecular basis of the inhibitory action of 2-deoxyglucose on glycosylation of protein is the incorporation of 2 deoxyglucosyl residues during early phases of the biosynthesis of the lipid-linked oligosaccharides. [ABSTRACT FROM AUTHOR]

The covalent-linked lipoprotein was isolated from purified peptidoglycan sacculi of early stationary phase Proteus mirabilis cells after digestion with Chalaropsis lysozyme and gel filtration. Only 1.7 % of the peptidoglycan subunits were found to be linked to lipoprotein. Further purification steps included chromatography on Sephadex G75 in 1.50% sodium dodecyl sulfate solution, removal of the detergent by acetone precipitation, and finally ultracentrifugation. The lipoprotein is soluble in water (about I µmol per mi) giving a slightly opalescent solution. Amino acid analysis in comparison with the covalent lipoprotein from Escherichia coli, kindly provided by V. Braun, revealed that firstly, the same set of amino acids is present except for methionine which is absent in the P. mirabilis lipoprotein, secondly the ratio of lipophilic and hydrophilic amino acids is essentially the same although P. mirabilis lipoprotein contains more acidic and fewer basic residues, thirdly, the P. mirabilis lipoprotein is composed of 60 amino acid residues as compared with 58 residues of the E. coli lipoprotein, fourthly, 55 To and 59 % of the lipoprotein molecules from E. coli and P. mirabilis, respectively, are linked to peptide crosslinked peptidoglycan dimers. Fatty acid analysis of the P. mirabilis lipoprotein gave 1.71 mol ester-linked and 1.14mol amide-linked (mainly palmitic acid) fatty acids per mol lipoprotein. Although the lipoproteins from E. coli and P. mirabilis have similar compositions and molecular weights they behave differently in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under various conditions. [ABSTRACT FROM AUTHOR]

1. The X-ray small-angle scattering of human plasma lipoprotein B of the low-density fraction (... = 1.016-1.060 g · cm-3) has been recorded to high precision at different electron density contrasts. 2. The overall structure of the particles is characterized by a quasi-spherical shape and radial symmetry. A maximum diameter of 23 nm and a molecular weight of 2.4 × 106 have been determined. 3. The internal structure is described in terms of a model consisting of spherical layers with different electron densities indicating that the neutral lipids are arranged in the core of the molecule up to a radius of about 8 nm surrounded by a monolayer of free cholesterol, phospholipids and protein. The neutral lipids are shown to be in an ordered, liquid crystalline state at 4 °C and to undergo a thermotropic transition into a disordered state at higher temperatures. [ABSTRACT FROM AUTHOR]

In an accompanying paper we describe a method to purify from very low density lipoproteins of egg yolk, two protein fractions (T1 and T2) with cofactor activity for lipoprotein lipases. When added to media containing (bovine milk) lipoprotein lipase and Intralipid (a lecithin-stabilized emulsion of soy bean triacylglycerols) they caused an immediate, several-fold increase in the rate of lipolysis. It has been considered characteristic for lipoprotein lipases that their activity is strongly suppressed by high concentrations of NaCl. Such inhibition was seen in assay systems with whole serum, lipoproteins, or crude fractions of egg apoproteins as source of cofactor activity. In contrast NaCl caused little or no inhibition in assay systems with no cofactor source added or with T1 or T2 proteins as cofactors, indicating that salt inhibition is not exerted directly on the active site of the enzyme or on its ability to interact productively with cofactor proteins. Addition of some of the other egg apoproteins changed the response to salt of activity stimulated by T1 and T2 proteins so that it became similar to that seen in assay systems with serum or other crude sources of cofactor activity. Thus, these other proteins could inhibit the enzyme activity and this inhibition was strongly potentiated by increasing the salt concentration of the medium. Inhibition at high salt concentration was seen also with hemoglobin and with myoglobin, suggesting that it is a rather nonspecific effect. It occurred immediately on addition of the inhibitory proteins to the medium, it was reversible and was relieved in a competitive manner by increased amounts of lipid substrate or of cofactor proteins. [ABSTRACT FROM AUTHOR]

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm²). At higher light intensities (above 25 mW/cm²) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 ± 5 photons per ATP molecule synthesized. According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions. [ABSTRACT FROM AUTHOR]

The purpose of this study was to establish a relationship between self-association and phospholipid binding of the human and the baboon apoA-1 protein. The enthalpy changes on binding dimyristoyl lecithin and lysolecithin to etther the human or the baboon native apoA-I protein were measured in a microcalorimeter. An endothermal process, most pronounced for the human apoprotein, was observed at low phospholipid levels. At higher phospholipid to protein ratios the binding was exothermal. Gel filtration experiments on Sephadex G-200 showed that the native apoprotein of both species consists of dimers and tetramers. The baboon native apoA-I protein contained a higher amount of dimers. After preincubation of the apoA-I protein with lysolecithin, the enthalpy changes measured on subsequent binding of dimyristoyl lecithin were shifted towards more exotherrnal values compared to the curve for the native apoprotein. The amplitude of this shift corresponds to that of the endothermal process observed on binding dimyristoyl lecithin to the native apoprotein. This process was attributed to a phospholipid-induced disaggregation of the apoA-I protein. Gel filtration data showed a decreased extent of aggregation in the apoA-I protein preincubated with lysolecithin. This sample consisted exclusively of dimers. Ultracentrifugal flotation of the complexes formed between the apoA-I protein, and respectively dimyristoyl lecithin and spbingomyelin indicated that preincubation with lysolecithin increased the extent of complex formation. These results suggest that the dimeric form of the apoA-I protein possesses the highest affinity for phospholipids. Any dissociation of higher polymers enhances the phospholipid-binding capacity of the human and the baboon apoA-I protein. [ABSTRACT FROM AUTHOR]

A direct method for determining the binding of tritiated water to lipids is described. The experimental conditions were practically identical to those previously employed (1974) in the determination of the cofactor activities of a series of oleyl-lipids in the reactivation of the C55-isoprenoid alcohol phosphokinase apoprotein. Active cofactor lipids (dioleyl lecithin, sodium oleate, 1-monoolein, 1-monomyristin) bound between 2.3 and 5.3 nmol ³H2O per nmol lipid, whereas less than 0.14 nmol ³H2O were bound per nmol of the inactive lipids (1,2- and 1,3-diolein, triolein, oleyl alcohol, methyl oleate, cholesteryl oleate). When exposed to ³H20 vapour, the active lipids adsorbed between 1 and 2 nmol ³H20 per nmol lipid, whereas the inactive lipids adsorbed less than 0.1 nmol ³H20 per nmol lipid. The active lipid cofactor, egg lecithin, bound more than twice as much ³H20 as egg phosphatidylethanolamine which was devoid of cofactor activity in the absence of detergent. Appropriately hydrated lipid polar groups are concluded to be required for an alignment with polar amino acid side chains of the enzyme apoprotein in the formation of a mixed micellar lipoprotein complex. The enzyme reaction might occur at the resulting lipoprotein/water interface. [ABSTRACT FROM AUTHOR]

Dinitrophenylated dipalmitoyl phosphatidylethanolamine and its lyso derivative have been shown to bind to the lipid-free ATPase protein derived from the sarcoplasmic reticulum. The binding of these lipids is accompanied by the quenching of up to 95% of the tryptophyl fluorescence of the protein. This effect is reversed by 9–I0 mM deoxycholate. The solubility of the lipid-free ATPase protein in the absence of deoxycholate and the solubility of submillimolar concentrations of the dinitrophenylated monopalmitoyl phosphatidylethanolamine anion in aqueous media allowed binding experiments using this lipid ligand to be carried out in a simple buffer system. It is shown that in the case of this lipid the initial phase of the binding process displays an apparent positive co-operativity. Data from the second phase in the saturation of the protein with this lipid is consistent with binding to independent, equivalent, non-interacting sites with a microscopic (intrinsic) association constant of 1.63 × 106 M&sup-1;, the fluorescence being quenched in a geometric fashion. Altogether a total of about 15 molecules of this lipid may be bound by the protein. [ABSTRACT FROM AUTHOR]

The cyanogens bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionineserine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largerly due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced. None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60%,of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto. [ABSTRACT FROM AUTHOR]

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 °C. The activity is a polynucleotide-pyrophosphorylatlng reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nucleic acids or polynucleotides as effectors. The product is not covalently linked to the effector. [ABSTRACT FROM AUTHOR]