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Determinations of the molcular weight by aid of gel filtration demonstrate that ferredoxin from Clostridium pasteurianum can exist as well in a monomeric (Mr = 6000) as in a dimeric form (Mr= 12000). The dimerisation takes place in the presence of oxygen. Using exactly anaerobic conditions pure monomeric material can be prepared; using aerobic conditions, however, pure dimeric material or a mixture of monomers, dimers, and apoferredoxins of higher molecular weights appear. Comparing the analytical data of the active center one finds eight atoms of iron and labile sulfur in monomeric ferredoxin, whereas four atoms of iron and labile sulfur are found in the subunit of dimeric ferredoxin. Results of the determination of the Hg-content in mersalyl-apoferredoxin prepared from a dimeric sample and of the titration of free SH-groups in monomers and dimers can be explained assuming that four cysteinyl residues per subunit participate in disulfide bridges in dimeric ferredoxin. That means, dimerisation is accompanied by a loss of iron and labile sulfur and therefore by a decrease of the absorption coefficient ε390 = 30000 M-l × cm-1 to ε390 = 15000 M-1 × cm-1 related to one subunit with the molecular weight of 6000. The ratio of absorbances A390: A280 seems to remain unchanged. In the phosphoroclastic system the biological activity of the dimeric ferredoxin decreases by a factor of two referring to one subunit. [ABSTRACT FROM AUTHOR]

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (≈98 %) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (≈97 %). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the N-terminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (KD = 1.28 nM), human/rat CRF (KD = 1.09 nM), urocortin (KD = 0.37 nM) and sauvagine (KD = 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (KD = 12.4 nM). In agreement with the binding data half maximum effective EC50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1. [ABSTRACT FROM AUTHOR]

DNA-bound polypeptide complexes composed of several non-histone polypeptides that resisted harsh DNA deproteinization procedures were characterized. The three major polypeptides of these complexes have molecular masses of 62, 52, and 40 kDa. They constitute supramolecular structures that reside on isolated DNA in dense clusters. The supramolecular complexes were released from DNA as globular 12.8±0.8-nm panicles: these particles were gradually disassembled to form smaller supramolecular structures. The DNA-bound complexes comprise of an encrypted adenosinetriphosphatase/phosphatase activity, which is a minor but intrinsic component of the complexes. The enzyme remained inactive as long as the complexes were bound to DNA. However, the enzyme was activated concomitantly with the progression of DNA digestion, which indicated that DNA was involved in the downregulation of the enzyme. The inactive DNA-restrained complex could not be restored in vitro, which indicated its nontrivial nature. Once released from DNA, the enzyme was inactivated over a period of several hours. However, in the DNA-associated complexes its potential to become activated during DNA digestion was conserved for several months. In the activated state, the enzyme showed an optimum activity at pH 9.5, was stimulated by Mg2+, inhibited by vanadate and EDTA, but was not significantly inhibited by okadaic acid. The active enzyme, which consists of two subunits of 56 kDa and 59 kDa, can be released from the supramolecular structures by agarose gel electrophoresis. A regulatory mechanism therefore exists for the downregulation of this phosphatase by DNA. [ABSTRACT FROM AUTHOR]

Ca2+ and Mg2- binding to rat parvalburnin was measured by means of the fluorescent Ca2+ indicator fluo-3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur J. Biochem. 202, 1333-1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35°C and pH 7.2 are 11.0 ± 1.8 nM and 41 ± 8 μM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo-3 are endothermic entropy-driven processes. [ABSTRACT FROM AUTHOR]

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase). MobB and MobC (Scherzinger, E., Lurz, R., Otto. S & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-481. In this study we report the purification of MobA to apparent bomogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by sing nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex. [ABSTRACT FROM AUTHOR]

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition C2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine α-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of α-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the α-lactalbumin-Ca2+ complex in 50 mM Hepes containing 150 mM Na2+ at pH 7.4 and 25 °C, was found to be 123 ± 2 nM and 103 ± 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to α-lactalbumin did not depend on pH in the range 6.6–8.4 and was differently affected by Na2+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na2+ at pH 7.4, was lowered to 119 ± 13 nM and the number of Ca2+ bound/molecule α-lactalbumin was lowered to 0.069 ± 0.006. No interaction between fluo-3 and α-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements. [ABSTRACT FROM AUTHOR]

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell cybernetic aspects. [ABSTRACT FROM AUTHOR]

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/²-H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40 -45'C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A. [ABSTRACT FROM AUTHOR]

Type VIIi collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochioride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen. [ABSTRACT FROM AUTHOR]

Ribonuclease T1 was studied by two-dimensional ¹H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequencespecific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and Cβ protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects. [ABSTRACT FROM AUTHOR]

DNA (cytosine-5-)-methyltransferase was purified as a single polypeptide (190 kDa by SDS-PAGE) from mouse P815 mastocytoma cells. This enzyme transfers methyl groups to unmethylated as well as to hemimethylated DNA sites with a strong preference for the hemimethylated substrate. A structural analysis of the isolated enzyme by electron microscopical techniques was undertaken. On the basis of the results obtained, we propose a model for the enzyme structure. This model describes the enzyme as a hemi-elliptical globular structure with dimensions of 5.4-6.7 nm for the height h and 10.3 - 10.8 nm for the diameter d, respectively; this globular structure bears a small appendix at the flat side. A molecular mass of 235- 250 kDa is calculated from the measured dimensions. Limited trypsin digestion of the enzyme led to a 160-kDa fragment which preserved the gross morphology of the original material. The possible structure function relationships are discussed. [ABSTRACT FROM AUTHOR]

1. ATP γP-imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+-ATPase of sarcoplasmic reticulum in a biphasic manner. A fast first phase is concentration-dependent while a slower second phase is independent of the inhibitor concentration. 2. The inhibition is calcium- and magnesium-dependent. No inhibition occurs in the absence of either cation. 3. Inhibition of the Ca2+-ATPase can be prevented by the protection with ATP. 4. The loss of ATPase activity is pH-dependent. Maximal inhibition coincides with maximal ATPase activity. It indicates a participation of the reacting amino acid side chain in the catalytic cycle. 5. The incorporation of radioactive inhibitor is reversed in a time-dependent fashion while the Ca2+-ATPase remains inhibited. 6. We conclude that ATP γP-imidazolidate initially reacts covalently with an amino acid side chain, probably Asp-351, but is subsequently expelled by a reaction with a second amino acid. 7. This two-step reaction induces an intramolecular cross-link which can be shown by the creation of a new protein band on SDS-PAGE which originates in the Ca2+-ATPase. [ABSTRACT FROM AUTHOR]

Antibody specificity is limited, and different antigens may cross-react to varying degrees with the same antibody. An example is monoclonal antibody 42PF elicited against a 29-residue random-coil peptide. 42PF cross-reacts with a triple-stranded coiled coil of related amino acid sequence, and there was circumstantial evidence for an antibody-induced opening of the coiled coil [Leder, L., Berger, C., Bornhauser. S., Wendt. H., Ackermann, F., Jelesarov, I. & Bosshard, H. R. (1995) Biochemistry 34, 16509–16518]. To reveal in a direct way that antibody 42PF induces the opening of the coiled coil, we have compared the rates of H/D exchange of amide protons in the free and in the antibody-bound coiled-coil peptide using electrospray-ionization mass spectrometry for the analysis of deuterium incorporation. Complete H/D exchange lasted several days in the tightly folded coiled-coil conformation while in the presence of antibody 42PF the overall exchange took only minutes, corresponding to a thousand-fold decrease of protection of the most slowly exchanging amide hydrogens of the folded coiled coil. This demonstrates that the antibody induced a considerable opening of the coiled coil. [ABSTRACT FROM AUTHOR]

Chromatin of a high structural order was prepared by a method avoiding drastic mechanical stress, as well as strong changes of the ionic strength, and was investigated by small-angle X-ray scattering. The results obtained support the idea that the quaternary structure of completely hydrated chromatin in solution is a solenoid with a diameter of about 32-34 nm. Gel chromatography of this chromatin yields solutions containing mainly quaternary structures with differing portions of tertiary structure. Decrease of the ionic strength results in an increase of loosening and unfolding of quaternary structures. Reconstitution by readjustment of the physiological ionic strength indicates slight differences between reconstituted and original higher-order structures. [ABSTRACT FROM AUTHOR]

Activity of pseudocholinesterase (acylcholine-acyl-hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholin- esterase of the afflicted members of both families (male and female) varied between 4800 U/I and 10200 U/I (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine. [ABSTRACT FROM AUTHOR]

The kinetic properties of two of the partially separated isoenzymes I and V of pig liver esterase were studied. The cholinesterase-like isoenzyme I hydrolyses butyrylcholine as well as various other esters and aromatic amides. This isoenzyme is sensitive to 0.01 mM physostigmine and to fluoride. The second type (isoenzyme V) has the features of the so-called aliesterase: it acts preferentially on short-chain aliphatic esters, does not hydrolyse butyrylcholine and has only low activity towards amides. Further differences exist with regard to sensitivity towards organophosphorous inhibitors, to the influence of organic solvents and to the pH optimum. Transacylation reactions with methanol as an acceptor are mainly catalyzed by the isoenzyme of the aliesterase type. The esterase forms III and IV, which are located between isoenzymes I and V on the isoelectric focussins column, show kinetic features similar to those of a mixture of I and V. A stepwise increase or decrease, respectively, of certain kinetic properties, e.g. specific activities, is observed for the sequence I, III, IV, V, A quantitative comparison of the kinetic properties supports our proposed subunit model [1], according to which the main components of these four esterase fractions are the trimers γγγ, αγγ, ααγ, and ααα. These results offer explanations for many of the very complex and often controversial results formerly obtained with the heterogeneous pig liver esterase. [ABSTRACT FROM AUTHOR]

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis( 4-nitro-[14C] phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals. [ABSTRACT FROM AUTHOR]

Polysomes specifically synthesizing the large subunit of ribulose-l.5-bisphosphate carboxylase were isolated from Chlamydomonas reinhardi cells by the indirect immunoprecipitation method. Electrophoretic analysis showed that the immunoprecipitated polysomes were of chloroplast origin. The mRNA coding for the large subunit which was purified from immunoprecipitated polysomes migrated at the 19-S position on sucrose density gradients, and its molecular weight was estimated to be 7.3 ×105 by acid-urea/agarose gel electrophoresis. The mRNA was translated in vitro with a cell-free protein-synthesizing system derived from Escherichia coli to give full-length large-subunit polypeptides. [ABSTRACT FROM AUTHOR]

Gel chromatography indicates that putidamonooxin has a molecular weight of about 126000. On the other hand, the amino acid composition and the iron-to-protein ratio point to a minimal molecular weight of 33000 and 31000 respectively. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme migrated as a homogeneous band corresponding to a molecular weight of about 40000. The number of spots found in the tryptic peptide map of the carboxymethylated and digested enzyme indicates that putidamonooxin is composed of three or four identical subunits. After covalent crosslinking of the subunits with dimethyl suberimidate and subsequent dodecylsulfate electrophoresis the main bands were in the molecular weight range of 40000, 87000 and 124000. These findings lead us to propose that putidamonooxin is either a trimer or tetramer. The amino acid composition of putidamonooxin and related data calculated from this are given. The isoelectric point was shown by isoelectric focusing to be at pH 4.7. Low-temperature optical spectra of the reduced and oxidized enzyme as well as of three different putidamonooxin, substrate complexes are given together with those recorded at 10° C. Enzyme-substrate binding spectra are observed with the oxidized putidamonooxin but not with the reduced enzyme. For the oxidized putidamonooxin a molar absorption coefficient at 455nm of 14.7mM-1 cm-1 was determined, Ks values of putidamonooxin towards different substrates and substrate analogues (i.e. tight couplers, partial uncouplers and uncouplers) are presented and possible reasons for the difference between the Ks values here obtained and the previously reported Km values are discussed. [ABSTRACT FROM AUTHOR]

The T 7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initiate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involves the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3' : 5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CTP are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initiators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2-4-fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from initiation of DNA chains tie novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA- binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with φX174 DNA as template leads to the generation of 'rolling circle-dike' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initiations can occur tie novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo. [ABSTRACT FROM AUTHOR]

Proton nuclear magnetic resonance shifts, spin-lattice and spin-spin relaxation times have been measured of low-spin bis-pyridine ferriprotoporphyrin(IX) dimethyl ester in chloroform. From the relaxation behavior the hyperfine coupling constant has been obtained and the contact term of the chemical shift was calculated. Deviations between measured and calculated chemical shifts may be attributed to second-order Zeeman interactions. The geometry of pyridine coordinated to the fifth and sixth position of ferriprotoporphyrin(IX) dimethyl ester was estimated from measured relaxation ratse. From the non-exponential decay of the Mz magnetization a mean lifetime of &taub = 50 ms for pyridine attached to low-spin ferriprotoporphyrin(IX) dimethyl ester was found at 253 K. [ABSTRACT FROM AUTHOR]

The main molecular and catalytic properties of an acetanilide-hydrolyzing enzyme from Pseudomonas acidovorans AE 1, purified to a homogeneous state, were investigated. The molecular weight was 57500 as determined by gel filtration and 55300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56700 was obtained. Based on the reaction of the highly purified enzyme with diethyl-4-nitrophenyl phosphate an equivalent weight of approximately 59100 was found. From these results it was concluded that the enzyme consists of a single polypeptide chain and contains one active site per molecule. The enzyme hydrolyzed esters as well as certain aromatic amides. It also catalysed the transfer of acetyl groups to phenetidine yielding phenacetin. The activities towards aliphatic esters were much smaller. The enzyme was stable at pH values ranging from 7 to 9 and its pH-optimum was about 10. It was strongly inhibited by organophosphorous compounds, like diethyl-4-nitrophenyl phosphate or diisopropylphosphorofiuoridate, as well as by physostigmine sulfate and - SH-blocking reagents, like HgCl2 or 4-chloromercuribenzoic acid. o-Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition. [ABSTRACT FROM AUTHOR]

The initial rate of incorporation of 14C or ³H-labeled choline into Ehrlich-Lettré ascites cells of the glycogen-free strain seven days after incubation was investigated in vitro. 1.At choline concentrations in the medium between 6 to 30 μM and 100 to 500 μM the choline uptake by the cells followed Michaelis-Menton kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10-values of 2.1 at the high and of 2.4 at the low choline molarity. The Km-values increased from 27 μM to 58.8 at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15 °C and 37 °C. Arrhenius plot of the V value gave two lines, one with a transition temperature at 25°C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol. 2. It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier-mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose, or 2,4-diitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4-dinitrophenol and p-chloromercuribenzoate. 3.Choline incorporation into the acid-insoluble material (lecithin) gave linear Michaelis-Menton kinetics at the low and the high substrate concentration respectively. Km-values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier-mediated process was in the order: mitochondria (50%) > plasma membranes (25%) > nuclei (14%) > microsomes (9%) > supernatant (1.5%). 4. Treatment of the cells with p-chloromercuribenzoate or heat shock at 50 %deg;C markedly reduced the choline uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p-chloromercuribenzoate was competitive and that of the heat shock non-competitive in nature. Further the choline uptake by the cells was found to be the rate-limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool. [ABSTRACT FROM AUTHOR]

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zürich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of ΔH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to AH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-α chains and NO-Hb Zürich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-β chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-β chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the α chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened σ-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure. [ABSTRACT FROM AUTHOR]

Phage T7 DNA polymerase in cooperation with T7 RNA polymerase can replicate φX174 singlestranded circular DNA. For optimal DNA polymerizing activity, the reaction requires a mixture of all four ribonucleoside triphosphates, or ATP and GTP, or high concentrations (1.5 mM) of ATP alone, in addition to the four deoxyribonucleoside triphosphates. With ATP as the only ribonucleotide present, T7 RNA polymerase is able to carry out φX174 DNA-directed synthesis of poly(adenylic acid) de novo. The poly(A) chains formed serve as a primer for T7 DNA polymerase. The DNA synthesized under conditions of coupled poly(A-DNA synthesis is complementary to the template DNA and contains poly(A)sequences covalently attached to it. With Escherichia coli unwinding protein complexed to the template DNA, the poly(A) or RNA-primer synthesis is strongly inhibited. This is in marked contrast to the stimulatory effect of the unwinding protein on the action of T7 DNA polymerase. [ABSTRACT FROM AUTHOR]

The electron paramagnetic resonance (EPR) line shape of Mn[SUP2+] ions bound in crystallized concanavalin A been analayzed at two microwave frequencies, 9 and 35 GHz. From a comparison between measured and simulated EPR-spectra, the correlation times for the process modulating the zero field splitting (zfs) interaction and the magntiude of the zfs-parameters have been determined. In the absence of Ca[SUP2+] ions a correlation time of 10 ps (at 4 °), typical for the thermal motions of the diffusing transition meal ions, has been determined. In the presence of Ca[SUP2+] ions the Mn[SUP2+] -spectrum of conconavalin A reveals forbidden transitions (Δm = ± 1), indicating that the mean, life-time for the transition meal ions at the binding site S1 is increased (τ[SUBass] &rsim; 10 ns) under the action of calcium ions bound at S2. [ABSTRACT FROM AUTHOR]

Ferredoxin from Clostridium pasteurianum which is characterized by two clusters containing four sulfur-linked iron atoms each, was studied by Mößbauer and electron spin resonance spectroscopy. The iron of oxidized ferredoxin is high-spin Fe(III) (S = 5/2) coupled antiparallel magnetically as concluded from the magnitude of the quadrupole splitting and its slight temperature dependence. Thus the net spin of the cluster is S = 0. This explains why no magnetic hyperfine splitting was observed in the Mößbauer absorption spectrum of oxidized ferredoxin at 4.2 K. The observation of at least two quadrupole doublets with a ratio of intensities deviating from unity points to a slight inequivatence of the ligand field symmetry of the individual iron atoms. Reduction of oxidized, ferredoxin leads to an uptake of one electron per cluster. The net electron spin of each cluster of reduced ferredoxin is thus S = ½. Spin-spin coupling of both the clusters prevents the appearance of a magnetic hyperfine splitting at low temperature. In an external magnetic field of 20 kG the coupling is removed and a hyperfine structure is observed. Under the conditions of X-band electron spin resonance spectroscopy, i.e. in a magnetic field of 3.5 kG, the spin-spin coupling is not totally removed. Therefore, an anisotropic 7-line spectrum originating from a system with S = 1, is observed. Under chemical conditions producing partly reduced ferredoxin a 2-line spectrum from the uncoupled S = ½ systems is also found. It indicates axial symmetry of the innermolecular electric field, Mößbauer spectroscopy enabled the detection of various amounts of non-cluster iron in samples of oxidized and reduced ferredoxin. [ABSTRACT FROM AUTHOR]

The light-stable tobacco phytochrome apoptrotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct. The spectral properties of the reconstituted PHYB-phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies. The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far-red-light-absorbing (Pfr) form and 356 nm and 658 nm for the red-light-absorbing (Pr) form. The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB-phycocyanobilin adduct with the spectral properties of the Pr form. Kinetic analyses of the in vitro reconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first-order rate constant of 2.8 × 10−²s−¹. The similarity with the reported rate constant for the reconstitution of light-labile phytochrome (PHYA) from oat [Li, L. & Lagarias, J. C. (1992) Phytochrome assembly. J. Biol. Chem. 267, 19 204–19 210] suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB. [ABSTRACT FROM AUTHOR]

Examines the structure of DNA junctions and their interaction with enzymes. Cruciform structures formed by inverted-repeat sequences in supercoiled DNA; Attempts to model the structure of a four-way junction in DNA; Analysis of the structure of the four-way junction by fluorescence resonance energy transfer and gel-electrophoretic analysis.

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cellfree extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes. [ABSTRACT FROM AUTHOR]

In barley seedlings grown in the dark large amounts of thionin-specific mRNAs are present, the concentration of which rapidly declines once the seedling is exposed to light. This rapid light effect is mediated by a complex interaction of possibly two photoreceptors, phytochrome and a blue-light-absorbing photoreceptor. Parallel to the decline in mRNA content, the de novo synthesis of leaf-specific thionins ceases rapidly upon illumination of etiolated seedlings. However, thionins which have accumulated before the onset of illumination remain stable within the seedling at high concentrations. In younger leaves of mature, nonstressed barley plants grown under a 16-h-light/8-h-dark cycle thionins are still present, although at much lower concentrations. In these plants, synthesis and accumulation of thionins occur predominantly in the meristematic zone at the leaf basis, which is shielded from light through the sheath of the preceding leaf. In mature light-adapted barley plants, mRNA encoding leaf-specific thionins may reaccumulate if these plants are exposed to pathogens or other stresses. Thus, the inhibitory effect of light on the biosynthesis of thionins may be overruled by stress- and pathogen-induced signals. [ABSTRACT FROM AUTHOR]

Ribonuclease T1 was studied by two-dimensional ¹H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequencespecific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and Cβ protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects. [ABSTRACT FROM AUTHOR]

Hepatic-type fatty-acid-binding protein (hFABP) from the cytosol of bovine liver is a 14.4-k Da neutral protein with a blocked N-terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 µM and 2.15 µM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12% α-helix, 45% β-structure, 15% β-turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as ¹H-NMR analysis of His-C2 and His-C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyro-carbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From ¹H-NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/ protein interaction. [ABSTRACT FROM AUTHOR]

The transcriptional rates of four different genes in shoots of barley grown under different light regimes were quantified by monitoring nuclear RNA transcripts using gene-specific hybridization probes. Isolated nuclei were pulse-labelled with [α-32]UTP and the relative rates of light-harvesting chlorophyll a/b protein (LHCP) mRNA, NADPH:protochlorophyllide oxidoreductase mRNA, B1 bordein mRNA, and 26-S rRNA synthesis were measured. Irradiation of dark-grown plants with a red light pulse increased the rate of LHCP mRNA synthesis tenfold within 3 h, and the rate of rRNA synthesis more than twofold within 9 h. The relative rate of synthesis of the oxidoreductase mRNA decreased following a red light pulse reaching a minimum after 3–6 h. As a direct proof of phytochrome involvement in the light-induced stimulation of LHCP and the repression of the oxidoreductase transcripts for both responses, red/far-red reversibility could be demonstrated. We conclude that phytochrome is able both to increase the transcription of certain nuclear genes and decrease the transcription of others. [ABSTRACT FROM AUTHOR]

Secretion of hemopexin (20% carbohydrate) and its dependence on glycosylation was studied in primary rat hepatocyte cultures in comparison to the secretion of transferrin (5% carbohydrate). 1. In pulse-chase experiments with [35S]methionine half of the labeled hemopexin was secreted in 30 min. By contrast, it took approximately 50 min for secretion of half of the transferrin. 2. Tunicamycin treatment of cultures significantly delayed the secretion of hemopexin but not that of transferrin. 3. During the pulse period a prominent intracellular precursor of hemopexin, smaller than the mature protein, was evident. It is concluded that the extent of glycosylation of a secretory protein is not necessarily a determinant of the transit time required for intracellular processing and secretion. In the case of hemopexin the glycosylation apparently facilitates the secretion although it is not an absolute prerequisite for the exocytosis of this protein. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS, endotoxin) of Gram-negative bacteria are among the main causes of sepsis and septic shock. In the present study, the influence of temperature on the biological activity of LPS was investigated. Lowering the temperature from 37 +C to 34.5 +C or to 30 +C significantly enhances in vitro tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 release induced by different LPS chemotypes and heat-inactivated Escherichia coli. This cytokine-increasing effect of lowering the temperature is highly mediated by serum proteins, particularly by LPS-binding protein (LBP) and low-density lipoproteins (LDL). In contrast, cytokine production induced by the superantigen toxic shock syndrome toxin-1 (TSST-1) from Gram-positive Staphyloccoccus aureus decreases by around 70 % at 30 +C as compared with 37 +C, corresponding to the expected effect of change in temperature and regardless of the presence of serum proteins. In order to explain the unexpected biological hypothermia effect with regard to LPS, the fluidity state of the lipid A portion of LPS as one important physico-chemical property possibly involved was investigated. The fluidity, determined by fluorescence polarization measurements, was found to decrease with decreasing temperature. These data suggest that a low fluid LPS chemotype is biologically more active than a more fluid one (and vice versa). Statistical analysis of the results shows a strong correlation between cytokine secretion and fluidity state of a given LPS chemotype (0.71 < r < 0.89, all P < 0.01). As a clinical consequence, these data may be one possible explanation for the higher mortality rate of hypothermic Gram-negative sepsis. [ABSTRACT FROM AUTHOR]

A yellow chromophore has been isolated from Avena coleoptiles grown in the dark. It had previously been shown by Zenk[zenk, M.H.(1967)Z.Pflanzenphysiol.56 ,122 -122-140] to be a flavin of still unidentified structure, and had been suggested to be "blue light' photoreceptor in this organism. The structure of this flavin (F1-X) has been obtained synthetically. The 5' malonylester linkage is relatively labile towards hydrolysis and photolysis, it flavin (absorption and florescence) are practically identical to those of normal riboflavin (vitamin B2. [ABSTRACT FROM AUTHOR]

Defective luteinizing-hormone-mediated camp generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25-dihydroxycholecalciferol (1,25-dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25-dihydroxycaliol receptor in the rat testis. Testes of non-rachitic, rachitic or acutely uremic male Sprague-Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25-[³H]-dihydroxycalciol (with or without 200-fold molar excess of unlabeled 1,25-dihydroxycalciol, subjected to 5-20% sucrose density-gradient separation (centrifugation with 255000 x g, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule migrated ar 3.5 S and could clearly be distinguished from the 6-S tissue-binding site for 25-hydroxycholecalciferol. The 1,25-dihydroxycalciol receptor was present in non-rarchitic and acutely uremic rats. Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25-dihydroxycalciol » 25-hydroxycholecalciferol > 1 α-hydroxycholecalciferol > 24(r),25-dihydroxycholecalciferol), low capacity (Nmax = 82 fmol/mg protein) and an equilibrium constant of Kd = 2.6 x 10-10 M. The receptor is protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N-ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25-dihydroxycalciol Binding of 1,25-dihydroxycalciol protects the receptor against inactivation with N-ethylmaleimide. [ABSTRACT FROM AUTHOR]

Human placenta is surprisingly rich in post-proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six-step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3–4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis. whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments. The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post-proline dipeptidyl peptidase from other sources. Phenylalanylprolyl-β-naphthylamide (Km = 0.02 mM, V = 92 U/mg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N-terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N-terminal sequence are cleaved. However, X-Pro-Pro-… structures, e. g. as in bradykinin, are not attacked. 1 mM bis-(4-nitrophenyl)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn&sup2+; and by other heavy metals. [ABSTRACT FROM AUTHOR]

The self-stabilization of the energy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase. The experiments were performed in a stirred flow-through reactor containing gel-entrapped enzymes. The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution or the flow rate through the react ion chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states. [ABSTRACT FROM AUTHOR]

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit. X-rays are diffracted to 0.35-rim resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase. [ABSTRACT FROM AUTHOR]

The properties of proteolytically non-modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be s200.w = 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found. The partial specific volume of the enzyme as determined by three different methods at 20 °C is v2 = 0.742 ml/g. From equilibrium sedimentation a molecular weight of 835000 + 32000 was calculated for the native enzyme. Amino acid analysis of the enzyme was performed and the number of half-cystine residues determined as 11⁄100000g is in good agreement with the number of titratable -SH groups after denaturation of the enzyme. The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15°/, smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate. Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered. [ABSTRACT FROM AUTHOR]

The Ca-transport system of sarcoplasmic vesicles of rabbits is altered by experimental uremia. 1. The influx rate constant of the experimental membranes decrease with a resulting decrease of the calcium influx rate. 2. The experimental membranes transport a smaller amount of Ca2+ per mol of ATP split than the controls, i.e. their transport ratio is decreased. 3. The calcium permeability of the experimental membranes increases with a resulting decreased concentrating ability. 4. The phosphatide content but not the cholesterol content of the experimental membranes de- creases with a consequent increase of the cholesterol/phosphatide ratio. 5. The fatty acid pattern of total phosphatides of the experimental membranes changes. A relative decrease of palmitic acid and oleic acid occurs and a relative increase of stearic, arachidonic and higher unsaturated fatty acids. 6. The altered lipid composition of the membranes does not change the temperature dependence of the kinetics. [ABSTRACT FROM AUTHOR]

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.02–1.7 μg/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S]0.5 is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations. Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer (Mr 190000) and active multimers of the dimeric form are involved. The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer. [ABSTRACT FROM AUTHOR]

Yeast phosphofructokinase having a molecular weight of 750000-800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits α and β (Mr 120 000) are converted to α' and β' (Mr approximately 90 000), and in the second step, accompanied by a decrease in enzyme activity, α' is converted to α' (Mr 80 000) and two further fragments having Mr 45 000 and 35 000 become detectable. In the course of this conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of α' and β', whereas ATP prevents only degradation of β'. When in presence of ATP α' is degraded to α'', the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of this molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of α'' to β' is one-to-one. [ABSTRACT FROM AUTHOR]

Parameters of plasma free fatty acid metabolism (pool size, half time, disappearance rate, turnover time and absolute turnover rate), the influx of plasma free fatty acids into the glycerides of brown adipose tissue and the pathway of triglyceride synthesis in brown adipose tissue (glycerol-1-phosphate versus monoglyceride pathway) were examined after intravenous injection of [1-14C]palmitate in newborn rabbits. In the thermoneutral environment of 35 °C the turnover rate of plasma free fatty acids was 10.20 µmol/min per 100 g body weight and its flux into the glycerides of brown adipose tissue 0.367 µmol/min per 100 g body weight. Cold exposure at an ambient temperature of 20 °C caused a decrease to 5.84 µmol/min and 0.207 µmol/min per 100 g body weight, respectively. Both under basal conditions at an ambient temperature of 35 °C and under cold-induced thermogenesis at an ambient temperature of 20 °C triglyceride synthesis in brown adipose tissue ran through the glycerol 1-phosphate pathway. [ABSTRACT FROM AUTHOR]

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 °C and -60 °C. For the purine ribosides the ayerage activation energy was found to be 4.7 ± 0.5 kcal · mol-1 (20 ± 2 kJ · mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5′-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5′) are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5′-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine. [ABSTRACT FROM AUTHOR]

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH-value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6-phosphate or fructose 1,6-bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration. [ABSTRACT FROM AUTHOR]

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps. Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity. [ABSTRACT FROM AUTHOR]