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Presents a list of articles for publication in future issues of the "European Journal of Biochemistry". Conformation of physalaemin; Association of cross-linked proteins with a specific mRNA in the cytoplasm of HeLa cells; Transport of proteins into chloroplasts.

The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

This article presents a list of some of the forthcoming papers in biochemistry to be published in this journal. Some of the papers listed are "The Organization of the Chloroplast DNA in wheat and maize in the region containing the LS Gene," by B. Koller, H. Delius and T.A. Dyer, "Effect of Carbamination on the Buffering Power of Purified Human Hamoglobins A Solutions At Two Temperatures," by M. Castaing, E. Bursaux and C. Poyart, "The Complete Nucleotide of the Chicken Ovotransferrin mRNA," by J.M. Jeltsch and P. Chambon, "The Primary Structure of Hen Ovotransferrin," by J. Williams, T.C. Elleman, I.B. Kington, A.G. Wilkins and K.A. Kuhn.

1. Sequence analysis of the NADPH domain (residues 158 - 293) and of the interface domain (365 - 478) was based on 12 CNBr fragments, which were isolated using ion-exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid-phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. 2. In the NADPH domain, residue 197, which is involved in an induced-fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chainfold in other proteins. 3. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallographic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, Ile29, Leu34, Tyr13, Phe14, Lys34. His16, Arg17, and Trp3. From these data an mr of 2 × 51 600 was calculated for the FAD-free apoenzyme and an Mr of 2 × 52400 for the holoenzyme. [ABSTRACT FROM AUTHOR]

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

This paper is a further study of ion binding to protein surfaces and builds on the studies of the binding of [Crk(CN)6]3- and [Fe(edta)(H2O)]- previously reported [Williams et al. (1982) FEBS Lett. 15, 293-299; Eley et al. (1982) Eur. J. Biochem. 124, 295-303]. In the present paper the binding of polyaminocarboxylate complexes of gadolinium have been studied. Eight ion-binding sites have been identified on the surface of cytochrome c. These exhibit different binding specificities which, in some cases, are not fully understood. However it is clear that simple outer-sphere interactions are not the sole determining factor for the association of metal ion complexes with proteins. The NMR paramagnetic difference spectrum method has been shown to be good at locating binding sites and revealing qualitative differences in their relative affinities for a range of complex types. However the use of relaxation probes is not a good method for the quantitative determination of binding constants; for this, isostructural shift probes must be sought. [ABSTRACT FROM AUTHOR]

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysyl-endopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine- linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins. [ABSTRACT FROM AUTHOR]

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 °C, echistatin contains no α-helix but contains mostly β-turns and β-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 °C and 72 °C being irreversible. Raman spectra also indicate considerable β-turn and β-sheet (20%) structure and an absence of α-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 °C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either α-helical or β-sheet conformation. A number of turns could be characterised. An irregular β-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies. [ABSTRACT FROM AUTHOR]

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr = 64 500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heine substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in β subunits which, in solution, appears to have stronger contacts with the heine groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr ≤ 65 000). [ABSTRACT FROM AUTHOR]

In this paper we describe the reduction of corticosteroid metabolites containing the 17β-aldol side chain (isocorticosteroids) by aldose and aldehyde reductase from human tissues. Aldose reductase catalyzed the reduction of the aldehydes derived from cortisol and corticosterone at about the same rate, whereas aldehyde reductase preferentially acted on the aldehydes derived Irom 1 7-deoxycorticosteroids. At comparable rates of reduction the Michaelis constants for the best steroid aldehydes were one order of magnitude lower than for the hitherto best substrates. We propose that aldose and aldehyde reductase participate in the conversion of the corticosteroid ketol side chain to the glycol side chain via an aldol intermediate by the `long loop' pathway proposed by Monder and Bradlow [(1977) J. Steroid Biochem. 8. 897 - 908]. [ABSTRACT FROM AUTHOR]

The complete primary structure of the constant part of the μ-chain-disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. Two amino acid changes were found, at positions 309 (Ser ↵ Gly) and 333 (Val ↵ Gly) (GAL numbering). In two additional monoclonal n chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of μ chains exist in the human: (1) GAL type with Ser-309 and Val-333; (2) OU type with Gly-309 and Val-333; (3) SCO type with Ser-309 and Gly-333; (4) BOT/CO type with Gly-309 and Gly-333. The meaning of this molecular polymorphism is discussed. [ABSTRACT FROM AUTHOR]

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS 2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-I did not influence the binding of these factors in the absence of fMet-tRNA. However, in the presence of fMet-tRNA it was found that the enhancement of the fMet-tRNA binding by IF-1 was accompanied with an equimolar increase in binding of IF-2. Moreover, it appeared that also in absence of IF-I, fMet-tRNA binding is coupled with an equimolar enhancement of the IF-2 binding, which suggests the existence of a preribosomal complex between IF-2 and fMet-tRNA. The apparent K,, values for both the binding of fMet-tRNA and IF-2 to 30-S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF-I in dissociation and recycling of IF-2, described by others, and the stimulation on the 30-S subunit level might well be explained as pleiotropic effects of one basic action of IF-1, i.e. a conformational change of 30-S subunits. [ABSTRACT FROM AUTHOR]

The Thr-102 mutant of yeast iso-l-cytochrome c is a useful system for the study of structure-function relationships in this important class of electron transfer proteins, but little is known about its structure. Furthermore, few assignments of individual amino acid residues in yeast iso-l-cytochrome c have been made by proton NM R. Here we report assignments for nearly half of the amino acids in the reduced Thr-102 mutant of yeast iso1-cytochrome c. We also report assignments for the oxidized Thr-102 mutant. While the crystal structure of the reduced iso-l-cytochrome c (N. B. not the Thr-102 mutant) has been reported, there is currently little structural information concerning its solution structure and none concerning the oxidized protein. There is also no information concerning the structure of either oxidation state of the Thr-102 mutant. Comparison of the chemical shift and NOE data for the reduced Thr-102 mutant and comparison of paramagnetic shifts for analogous residues between this mutant and horse-heart and tuna cytochromes c reveal that both the basic fold of Thr-102 yeast isoI-cytochrome c and the region around the site of the mutation are the same as those found in the latter two proteins. It is concluded that the results from structure function studies using the Thr-102 mutant will be applicable to eukaryotic cytochrome c in general. This knowledge allows us to proceed to a description of some mutants of yeast iso-l-cytochrome c in the next paper. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms α, β and γ, differenciated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive β-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gone for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by λ540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1bx is the first membrane-bound form of pbp 1b able to bind labelled β-lactams, and is the precursor of pbp 1bβ which is, in turn, the precursor of pbp 1bγ. [ABSTRACT FROM AUTHOR]

The synthesis by yeast microsomes of compounds that are probably dolichol-pyrophosphate derivatives containing N,N'-diacetylchitobiose and several mannose residues is described. However, the presence of monosaccharide residues other than N-acetylglucosamine and mannose has not been ruled out. The amount of the lipid derivatives synthesized was enhanced by the addition to the incubation mixture of an organic-solvent-soluble extract from rat liver known to contain dolicholpyrophosphate oligosaccharides. Incubation of these derivatives with yeast microsomes led to the transfer of about fifteen percent of their saccharide moieties to endogenous proteins. The oligosaccharides released from the dolichol derivatives by mild acid hydrolysis could serve as primers for the synthesis of a polysaccharide having the characteristics of mannan outer-chain. It is suggested that the dolichol-pyrophosphate derivatives described in this paper are intermediates in the synthesis of mannan inner core. [ABSTRACT FROM AUTHOR]

Computer modelling has been used to investigate the effect of direct transfer of metabolites between consecutive enzymes (channelling) on the free concentrations of the channelled metabolites. When a channelled intermediate cannot participate in any other reactions, any increase in channelling tends to increase its free concentration, albeit very slightly, unless the increase in net flux brought about by the channel is compensated for by a simultaneous decrease in the activity of the route through the free intermediate, in which case channelling has no effect at all on the free steady-state concentration of the channelled intermediate. If the free intermediate is capable of participating in side reactions, channelling can decrease these side reactions, but only slightly unless virtually all of the final product results from flux through the channel and the rate constants for the direct pathway are virtually zero. In general, channelling appears not to provide a useful mechanism for maintaining intermediate concentrations at low levels. [ABSTRACT FROM AUTHOR]

In Bacillus subtilis, 5-azacytidine, an analog of cytidine, causes a time- and dose-dependent growth inhibition. Methyl donors are unable to overcome azacytidine-induced inhibition while pyrimidine nucleosides, except orotidine, can revert this inhibition totally. On the other hand, pyrimidine bases, except uracil, are unable to restore growth in azacytidine-treated cells. Uracil, at a high concentration, can revert growth inhibition only inefficiently. However, a considerable relief of growth inhibition by uracil occurs in the presence of a ribose donor. In azacytidine-treated B. subtilis cells methylation of bases in DNA is not affected either quantitatively or qualitatively and DNA methyltransferase activity remains unaltered as compared to the untreated cells, apparently due to the absence of azacytidine incorporation into the DNA. The inability of B. subtilis cytidine kinase to phosphorylate azacytidine is the probable reason for this non-incorporation. Analysis of the enzymes of de novo pyrimidine biosynthesis has shown that orotidine monophosphate pyrophosphorylase is specifically repressed by azacytidine treatment. [ABSTRACT FROM AUTHOR]

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalyzing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatase-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 µM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo. [ABSTRACT FROM AUTHOR]

A simple theorem is derived relating the extent to which enzymes in a metabolic pathway control the steady-state concentration of metabolites to the kinetic properties of those enzymes. The theorem gives insight into the mechanism by which the concentration of a second messenger is controlled by the enzymes that form and degrade it, and provides an alternative to the ‘cross-over theorem’. [ABSTRACT FROM AUTHOR]

Incubation of whole lymphocytes with UDP-N-acetyl [3H]glucosamine used as the only precursor leads to the formation of dolichyl diphosphate [3H]chitobiose. DolPP-(G1cNAc)2, and dolichyl diphosphate N-acetyl- [3H]glucosamine, DolPP-GlcNAc. Although very few dolichyl diphosphate oligosaccharides are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N-acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DolPP-(GIcNAc)1-2 and the incorporation of the N-acetyl[3H] glucosaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP-(GIcNAc)1 -2 were utilized in two ways: either their transfer onto proteins or their degradation into water-soluble saccharidic material. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DoIPP-(GlcNAc)1 -2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP-mannose. This direct mannosylation of chitobiosyl-proteins may represent a second route for the N-glycosylation of proteins. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

As a step towards understanding the role of nuclear ribonucleoprotein particles (hnRNA · proteins) in the nucleocytoplasmic transfer of heterogenous nuclear RNA sequences destined to become the cytoplasmic messenger RNA, the phosphorylation in vivo of the protein moiety of these hnRNA · proteins from HeLa cells has been studied. After exposure of HeLa cells to [32P]orthophosphate in vivo, purified hnRNA · proteins were found to contain radioactivity covalently bound to proteins in the form of serine and threonine phosphate esters. The association of these phosphoproteins with hnRNA was demonstrated by sedimentation analysis in sucrose gradients, isopyenic banding in cesium chloride density gradients and chromatography on oligo(dT)-cellulose. The pattern of radioactive phosphorylated species has been analysed by electrophoresis on sodium dodecylsulphate/polyacrylamide slab gels followed by autoradiography. Although some hot- trichloroacetic-acid-resistant 32P radioactivity was present throughout the gel, the highest amount of label was found associated with four discrete bands of molecular weights 28000, 30000, 37000 and 52000. Incorporation of 32P into hnRNA proteins could be detected after labeling periods as short as 15 min, the same species being labeled after 1 and 24-h labeling times. Although all the above species appear to be present in hnRNA · proteins of different size classes, as resolved by sedimenta- tion through sucrose gradients, their relative proportion is not reproducibly constant. Exposure of hnRNA · proteins to conditions of increasing ionic strength led to the ready disso- ciation of the 28000-Mr species at variance with other labeled species which appeared to be more firmly bound to hnRNA than the bulk of proteins. Upon treatment with pancreatic and T1 ribonucleases, the 28000-Mr species was also preferentially released while the 37000-Mr one was the main phosphorylated species remaining associated with core structures containing an RNA component resistant to nucleases which is now under further characterization. [ABSTRACT FROM AUTHOR]

Antibodies elicited by a synthetic immunogen, (Glu60Ala40)n -[Ala-DGlu(Lys-DAla-DAla)]5.1, related to a major class of peptidoglycan precursors, were purified by an affinity column of Sepharose 4B, to which the hapten, Ala-DGlu(Lys-DAla-DAla), was covalently attached and were then coupled to Sepharose 4B. Low-molecular-weight [14C]alanine-labeled products secreted by Micrococcus luteus cells grown in the presence of penicillin G were applied to the antibody-coupled gel. The bound material, which should be univalent to the antibody, was eluted completely by 0.02 mM α-tert-butyl- oxycarbonyl-lysyl-D-alanyl-D-alanine. High-molecular-weight uncross-linked soluble peptidoglycan secreted by M. luteus cells grown in the presence of penicillin G was completely bound to the affinity column, regardless of whether the soluble peptidoglycan was labeled in its alanine moieties or in its glucosamine and muramic acid residues A minor fraction of the bound soluble peptidoglycan was released with 0.02 mM tripeptide (‘paucivalent’ fraction). The remainder of the bound material was eluted with 2 mM tripeptide, indicating that a major portion of the high-molecular-weight material was ‘multivalent’. When glycan-labeled soluble peptidoglycan was stored in buffered saline at -5 °C for several months, a fraction of the labeled material was no longer bound to the affinity gel. Digestion of this non-binding fraction, as well as the paucivalent and multivalent fractions with hen egg-white lyso- zyme, was consistent with the affinity chromatographic data. These results indicate that an amidase present in these preparations cleaved different amounts of peptide from the soluble peptidoglycan preparations, producing strands which are polydisperse with respect to their peptide substitution. [ABSTRACT FROM AUTHOR]

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobic- hydrophilic interchanges in the sequence 51–55, which forms part of an α-helix in bovine ribo-nuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62–64). The enzyme differs at about 35–40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found. [ABSTRACT FROM AUTHOR]

1. Different types of bacteriorhodopsin chromophores and their reactions are described. This includes intermediates of the reconstitution reaction of the purple complex, intermediates of the photochemical cycle and a photochemically active 500-nm chromophore. In contrast to the native chromophore (purple complex) some of these species are reducible by borohydride yielding stable retinyl proteins. 2. Hydrolysis and/or extraction of the retinyl proteins reveals that in the corresponding non reduced parent chromophores the retinal is bound either noncovalently or covalently to lysyl residues of the polypeptide chain. 3. Retinol, retinyl lysine and their retro isomers isolated from the various reduced chromophores are identified by thin-layer chromatography and mass spectrometry. 4. The absorption spectra of the retinyl proteins and binding studies with retinol and retinal indicate that the cyclohexene ring and the side chain of the retinyl moiety are forced into a coplanar conformation by interaction with the protein. The three peaked absorption band of the reduced chromophores is due to this planarized conformation and not to a retro configuration of the retinyl moiety. 5. Isomerization to retro compounds can be achieved by HCl as is known to be the case for retinyl compounds in solution. In addition photochemical isomerization is observed in the case of planarized retinyl moieties. Thus the native protein structure is responsible not only for a specific conformation of the retinyl moiety but also for its specific reactivity. [ABSTRACT FROM AUTHOR]

The synthesis of a class of proteins with affinity for denatured DNA has been studied in synchronized cultures of the established hamster fibroblast line NIL-1 and its virus-transformed derivative NIL-1-hamster sarcoma virus. It has been found that the synthesis of a DNA-binding protein (P6, molecular weight 50000) in synchronized untransformed NIL-1 cells follows a pattern different from that observed in the transformed cells. The protein is low in stationary cultures of NIL-1 and NIL-1-hamster sarcoma virus but increases after the cells are stimulated to grow, although the time of maximal P6 synthesis relative to cellular DNA synthesis is different in NIL-1 and NIL-1-hamster sarcoma virus. In contrast, the pattern of synthesis of two other DNA-binding proteins (P7 and P8′) is essentially identical in synchronized untransformed and transformed cells. P7 is very low in resting cultures of both types of cells, but greatly increases early after the ceils are stimulated to divide, while P8′ is high in stationary cultures and decreases slightly after the initiation of cellular DNA synthesis has occurred. [ABSTRACT FROM AUTHOR]

The primary structure of the most acidic (pI = 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N-bromosuccinimide cleavage of the protein. The amino acid sequence so obtained is considered in comparison with those know for other parvalbumins and for rabbit troponin-C. [ABSTRACT FROM AUTHOR]

The use of 13C-NMR spectroscopy in the establishment of lipid A backbone structure from lipopolysaccharide of Yersinia pseudotuberculosis has been described. The 13C-NMR spectra of degraded lipid A and its N-acetate were obtained. The assignment of signals was made by comparison with the chemical shifts for 13C-NMR spectra of glucosaminitol, N-acetyl-glucosaminitol and their β-1.4 and β-1.6 disaccharides. It was shown that lipid A backbone of the lipopolysaccharide in question consists of β.-1.6-linked glucosamine disaccharide [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiaeis a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease inS. cerevisiae.It is encoded by theCYM1gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption ofCYM1not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71–77)catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) fromStaphylococcus carnosushave shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 491HN-15N, and 251HN-1Hα residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation. [ABSTRACT FROM AUTHOR]

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp,Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. TheL. vannameihistone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition againstMicrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µmwhile a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. [ABSTRACT FROM AUTHOR]

The classical metabolic control theory[Kacser, H.&Burns, J.A. (1973)Symp. Soc. Exp. Biol.27, 65–104; Heinrich, R.&Rapoport, T. (1974)Eur. J. Biochem.42, 89–95.] does not take into account experimental evidence for correlations between enzyme concentrations in the cell. We investigated the implications of two causes of linear correlations: competition between enzymes, which is a mere physical adaptation of the cell to the limitation of resources and space, and regulatory correlations, which result from the existence of regulatory networks. These correlations generate redistribution of enzyme concentrations when the concentration of an enzyme varies; this may dramatically alter the flux and metabolite concentration curves. In particular, negative correlations cause the flux to have a maximum value for a defined distribution of enzyme concentrations. Redistribution coefficients of enzyme concentrations allowed us to calculate the‘combined response coefficient’ that quantifies the response of flux or metabolite concentration to a perturbation of enzyme concentration. [ABSTRACT FROM AUTHOR]

Poly(β-l-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains ofPhysarum polycephalumthat differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 × LU898) were increased by 40–50% while those of a high producer strain (M3CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2–9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium. [ABSTRACT FROM AUTHOR]

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexinV,was strictlyCa2+-dependent,whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone- like function which is Ca2+-dependent. [ABSTRACT FROM AUTHOR]

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of ‘sweet fingers’, i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential ‘sweet fingers’ of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37–47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the ‘wedge model’, recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett. 526, 1–3.]. [ABSTRACT FROM AUTHOR]

Understanding the sequence determinants of protein structure, stability and folding is critical for understanding how natural proteins have evolved and how proteins can be engineered to perform novel functions. The complexity of the protein folding problem requires the ability to search large volumes of sequence space for proteins with specific structural or functional characteristics. Here we describe our efforts to identify novel proteins using a phage-display selection strategy from a `mini-exon' shuffling library generated from the yeast genome and from completely random sequence libraries, and compare the results to recent successes in generating novel proteins using in silico protein design. [ABSTRACT FROM AUTHOR]

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts. [ABSTRACT FROM AUTHOR]

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the β-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for β-lactam binding, dd-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 m−1·s−1). Against a set of five α- and ε-substituted l-Lys- d-Ala- d-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for Nε-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an Nε-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2- l-Lys- d-Ala- d-Ala ( kcat/ Km) was bell-shaped, with p Ka values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other dd-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs. [ABSTRACT FROM AUTHOR]

A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preferenence for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors – amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa. [ABSTRACT FROM AUTHOR]

Selected members of' the genus Astragalus (Fabaceae) are known for their ability to accumulate high levels of selenium, mainly in the form of Se-methyl-selenocysteine. With the aid of cell cultures we have investigated the molecular basis for selenium tolerance of these plants. It is shown that cultured cells from a selenium-accumulating Astragalus species synthesize Se-methyl-selenocysteine in contrast to those of a non-accumulating species and do not unspecifically incorporate selenium into proteins. The purification and biochemical characterization of a selenocysteine methyltransferase from cultured Astragalus bisculatus cells is described, which does not accept cysteine `as a substrate. We propose that this enzyme plays a crucial role in conferring selenium tolerance. [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]

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-l allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of file birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen. [ABSTRACT FROM AUTHOR]

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coti RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shill assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C-terminus or from N-terminal residues 6–39. A peptide corresponding to amino acid positions 152–169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303–353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA-binding domain of the RecA protein. [ABSTRACT FROM AUTHOR]

The major components of neurofibrillary tangles (NFT) in Alzheimer's disease are bundles of paired helical filaments (PHF) which are primarily composed of highly phosphorylated tau proteins (PHF-tau). To further understand the mechanism of PHF accumulation in NFT, we examined the calpain-induced proteolysis of highly purified and primarily non-aggregated PHF and normal tau proteins with various contents of phosphate isolated born either fetal (F-tau) or adult human brain (N-tau). The extent of proteolysis was determined by decreases in tau immunoreactivity using Western-blot analysis and a panel of site-specific tau antibodies (Alz 50, Tau-2, Tau 14, Tau-1, AT8, B-11, AH-1 and PHF-1). We found that lull-size polypeptides of N-tau and F-tau were similarly and rapidly proteolyzed in vitro by calpain (calpain II, 3.3 units/mg protein) during a 10-min incubation at 30°C, and that then half lives (t½) were 1.5 mm and 1.8 min, respectively. A major calpain-generated fragment was a 45-kDa polypeptide derived from the C-terminal region of PHF-tau, which forms a core of fragments. The results suggest that the inaccessibility of potential calpain-digestion sites in the filament core could contribute to the resistance of PHF to calpain and subsequently lead to the accumulation of PHF in Alzheimer's disease. The results also suggest that hyperphosphorylation of tau may be marginally involved in the resistance of PHF to degradation by calpain. Ultrastructural examination revealed that, in contrast to previous studies with trypsin, calpain did not alter the morphologic appearance of filaments after incubation with calpain, the majority of PHF remained short and disperse and the number of PHF aggregated into NFT-like clusters was not significantly increased. The results suggest that the role of calpain in promoting the aggregation and clustering of filaments is limited. [ABSTRACT FROM AUTHOR]

The control and regulation of metabolic systems are determined by their responses to changes in the internal metabolites (the internal state) and parameters of the system. In many cases, the concentrations of the intermediates are constrained by moiety conservations, for example those requiring that all intermediate forms of any enzyme sum to the conserved total concentration of that enzyme. In this study, we show how responses to changes in the internal state are related to responses to changes in the total amounts of conserved moieties. The relationship between these two different measures of control leads to a generalisation of the connectivity theorems. The results have important implications for the study of a variety of phenomena such as metabolite (coenzyme) sequestration, group-transfer and channeling. The relationships we derive make it possible to determine the control features of these pathways. As an illustration, two examples are chosen. The first shows the effect of sequestration of substrate moiety while the second deals with the sequestration of the enzyme moieties and enzyme/enzyme interactions. [ABSTRACT FROM AUTHOR]

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl β-D-maltoside. Chlorophyll-binding proteins from photosystem Il were isolated either by ultracentrifugation on a sucrose gradient, or by fiat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43. 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem Il core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem-II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration. while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing. [ABSTRACT FROM AUTHOR]

Direct interaction between the C-terminal portion of dystrophin and dystrophin-associated proteins wax investigated. The binding of dystrophin to each protein wax reconstituted by overlaying bacterially expressed dystrophin fusion proteins onto the blot membranes to which dystrophinassociated proteins were transferred after separation by SDS/PAGE with the following results. (a) Among the components of the glycoprotein complex which links dystrophin to the sarcolemma, a 43-kDa dystrophin-associated glycoprotein binds directly to dystrophin. Although at least one of the binding sites of this protein resides within the cysteine-rich domain of dystrophin, a contribution of additional amino acid residues within the first half of the C-terminal domain was also suggested for more secure binding. (b) Two other proteins also directly bind to dystrophin. Their binding sites are suggested to reside within the last half of the C-terminal domain which is alternatively spliced depending on the tissue type. Previously, based on the enzyme digestion experiments, we showed that the binding site for the glycoprotein complex on dystrophin is present within the cysteine-rich domain and the first half of the C-terminal domain [Suzuki, A., Yoshida, M., Yamamoto, H. & Ozawa, E. (1992) FEBS Lett. 308, 154-1601. Here, we have extended this work and found that the region which is involved in interaction with the complex is widely extended to the entire length of this part of the molecule. On the basis of the present results, we propose a model of molecular architecture at the binding site for the complex on dystrophin. [ABSTRACT FROM AUTHOR]

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coli β-galactosidase, the 70-kDa constitutive heat-shock proteins and the 68-kDa dsRNA-dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock. The heat-induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70-kDa constitutive heat-shock proteins and the 68-kDa protein kinase are ATP-binding proteins but ATP depletion also considerably increases the aggregation of β-galactosidase to the nuclear pellets, although this enzyme is not known lo be an ATP-binding molecule. Insolubilization of all four reporter proteins occurs in ATP-depleted cells even at normal growing temperatures (37°C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular 'free' chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP. Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP-depleted cells and for the ability of mitochondrial uncouplers to trigger a heat-shock response in some cells. [ABSTRACT FROM AUTHOR]

The lipopeptidophosphoglycan from Trypanosoma cruzi is a glycosylated inositol-phosphoceramide isolated from epimastigotes at the stationary phase of growth (4–5 days); We have now purified two similar glycoinositolphospholipids (glycoinositolphospholipid A and glycoinositolphospholipid B) from epimastigotes after the second day of culture growth. [3H]Palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in glycoinositolphospholipid A and into ceramide in glycoinositolphospholipid B. The lipids were released by incubation with glycosylphosphatidylinositol-specific phospholipase C from Bacillus thuringiensis or by chemical methods: After alkaline hydrolysis, the lipids were analysed by GLC/MS. In glycoinositolphospholipid A the resulting lipids corresponded to l-O-hexadecylglycerol and palmitic acid. The ceramide components in glycoinositolphospholipid B are sphinganine, palmitic acid and lignoceric acid. The oligosaccharides could degraded by nitrous acid and further enzymic treatment showed that the two glycoinositolphospholipids isolated from T. cruzi share the common core structure of the glycosylphosphatidylinositol membrane anchors. The microheterogeneity was determined, as well as the substitution by galactose and was mainly in the furanose configuration as was previously described for lipopeptidophosphoglycan. However, methylation analysis indicated that 20% of the galactose is in the pyranose form. Both glycoinositolphospholipids mainly differ in the lipid moiety. [ABSTRACT FROM AUTHOR]