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This article presents information on forthcoming papers to be published in coming issues of the periodical "European Journal of Biochemistry." Some of the papers are: "Localization of Histone HS in the Subunit Organization of Chromatin Using ImmunoeIectron Microscopy"; "Presence of an Amino Acid Oxidase in Photosystem II of Anacystis nidutans"; "Aldoximes: Active-Site Probes of Alcohol Dehydrogenases"; and "Quinone Reductases of Higher Plants."

The article presents the forthcoming papers that will be published in the "European Journal of Biochemistry." Some of these articles to be published are "Purification and Characterization of Thermus Caldophilus GK24 DNA Polymerase," by J.H. Park, J.S. Kim, S.-T. Kwon and D.-S. Lee, "Lactotransferrin Binding to Its Platelet Receptor Inhibits Platelet Aggregation," by B. Leveugle, J. Mazurier, D. Legrand, C. Mazurier, J. Montreuil and G. Spik and "Does Escherichia Coli Possess a Second Critrate Synthase Gene?," by A.J. Patton, D.W. Hough, P. Towner and M.J. Danson.

The article presents information on the forthcoming papers and articles that will be published by the "European Journal of Biochemistry." Some of them are Ionic-strength-dependent substrate inhibition of the lysis of Micrococcus luteus by hen egg-white lysozyme by I. M.A. Verhamme, G. W. K. van Dedem and A. R. Lauwers, A maximum of two tryptophan residues in gene-32 protein from phage T4 undergo stacking interactions with single-stranded polynucleotides, Cell-cycle-related metabolic and enzymatic events in proliferating rat thymocytes.

Presents articles for publication in future issues of the "European Journal of Biochemistry". Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain; Progression of mammalian DNA replication intermediates to mature chromatin; Purification, characterization and induction of phenylalanine ammonia lyase in Phaseolus vulgaris.

Turnip isoperoxidase TP 7 had its hemin group removed and was cleaved by trypsin. The digest was fractionated by gel filtration and high-voltage paper electrophoresis and yielded 24 peptides, which counted for all 296 amino acid residues of the enzyme. Sequence analyses on the tryptic peptides and, when necessary, on their thermolytic derivatives were carried out by manual Edman degradation followed by dansylation or by quantitative amino acid analysis of thiazolinones convened by HI. The four disulfide bridges and the only site of carbohydrate attachment of turnip peroxidase 7 are located. The present analysis of tryptic peptides has been complemented by cyanogen bromide fragments cleaved by chymotrypsin as described in the accompanying paper. [ABSTRACT FROM AUTHOR]

Tryptic and chymotryptic digestion of peptide B, the N-terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: -Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys-Met-. This sequence supports earlier conclusions dragon from fluorescence studies of thioredoxin, which indica ted a close relationship between one or both tryptophan residues and the disulfide bridge of the active site. Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C-terminal fragment (peptide A). [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

A subset of the spin systems assigned in the ¹H NMR of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: (a) selective spin-lattice relaxation times; (b) the dependence of line widths on temperature and the concentration of manganous ion; (c) fluorescence quenching; and (d) titration behaviour. Criteria are developed for distinguishing surface and buried residues. The significance for the function of DNA biding proteins is discussed. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = √1-q⊃ is fulfilled. In this relation L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load, i.e. the irreversible ATP-utilizing processes in the cell, and q the degree of coupling of oxidative phosphorylation driven by respiration. Since during short time intervals L11 and q are constant whereas L33 fluctuates in the cell, oxidative phosphorylation would only rarely operate at optimal efficiency due to violation of conductance matching. This paper demonstrates that the reversible ATP-utilizing reaction catalyzed by adenylate kinase can effectively compensate deviations from conductance matching in the presence of a fluctuating L33 and hence allows oxidative phosphorylation to operate at optimal efficiency in the cell. Since the adenylate kinase reaction was found to buffer a thermodynamic potential, i.e. the phosphate potential, this finding was generalized to the concept of thermodynamic buffering. The thermodynamic buffering ability of the adenylate kinase reaction was demonstrated by experiments with incubated rat-liver mitochondria. Considerations of changes introduced in the entropy production by the adenylate kinase reaction allowed to establish the theoretical framework for thermodynamic buffering. The ability of thermodynamic buffering to compensate deviations from conductance matching in the presence of fluctuating loads was demonstrated by computer simulations. The possibility of other reversible ATP-utilizing reactions, like the ones catalyzed by creatine kinase and arginine kinase, to contribute to thermodynamic buffering is discussed. Finally, the comparison of the theoretically calculated steady-state cytosolic adenine nucleotide concentrations with experimental data from perfused livers demonstrated that in livers from fed rats conductance matching is fulfilled on a time average and that the degree of coupling corresponded to qpec = 0.97 permitting the most economic maintenance of a maximal output power of oxidative phosphorylation. For the case of livers from starved rats this analysis suggested that the degree of coupling corresponded to qfec = 0.95, permitting the most economic maintenance of a maximal net rate of ATP synthesis at optimal efficiency of oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

It was demonstrated that an enzyme system(s) extracted from etiolated seedlings of Stizolobium hassjoo catalyzed the conversion of L-dihydroxyphenylalanine into stizolobinic acid. α-amino-6-carboxy-2-oxo-2H-pyran-3-propionic acid, and stizolobic acid, α-amino-6-earboxy-2-oxo-2H-pyran-4-propionic acid, in the presence of NADP+ or NAD+ under aerobic condition. Enzymically synthesized radioactive stizolobinic acid and stizolobic acid isolated from the reaction mixtures were purified and confirmed to have constant specific radioactivities by cocrystallization with authentic samples. Maximal activity of the enzyme preparation was obtained by using an insoluble polyphenol adsorbent (Polyclar AT) and a reducing agent (araboascorbic acid) in the extraction medium and by subsequent fractionation of the extract with ammonium sulfate followed by Sephadex G-25 gel filtration. Catalytic activity of the enzyme preparation was more unstable under aerobic condition than anaerobic, Attempts to stabilise the enzyme activity were made by the use of many substances which are known to stabilise other enzymes or expected to arrest the inactivation. Evidence is provided in this paper that the previously proposed biosynthetic pathways of stizolobinic acid and stizolobic acid from dihydroxyphenylalanine proceeded in the cell-free system from etiolated seedlings of S. hassjoo. [ABSTRACT FROM AUTHOR]

1. The arginine-requiring mutant of Chlamydomonas reinhardi, isolated by Ebersold in 1956 and called arg-1, shows no activity of the enzyme acetyl-glutamate-reductase, for this reason we propose to call this mutant arg C1. By analogy with this we call the arginine-requiring mutant arg-2, isolated by Eversole in 1956, arg G2. 2. Under circumstances in which the synthesis of the last enzyme of the arginine pathway, the argininosuccinate lyase, shows strong repression and derepression, we could not find any regulation of the synthesis of the 2nd up to and including the 6th enzyme of this pathway. 3. A more detailed study on the 6th enzyme of the pathway, the ornithine transcarbamylase, also failed to show any regulation of the synthesis. The findings of 2 and 3 support our hypothesis on the regulation of the arginine biosynthesis in Chl. Reinhardi, which we presented in the preceding paper. [ABSTRACT FROM AUTHOR]

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study ofunnatural substrates containingd-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1–4) derivatives, Boc-Tyr-d-Ala-Phe-Gly-N2H2Ph and Boc-Tyr-d-Ala-Phe-Gly-NH2, which contained ad-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1–4) derivatives could be catalyzed by subtilisin with Boc-Tyr-d-Ala-OCH2CF3 as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1–2) derivative Boc-Tyr-d-Ala-N2H2Ph was catalyzed byα-chymotrypsin in different organic solvents andd-Ala-N2H2Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied. [ABSTRACT FROM AUTHOR]

This paper presents the first purification of the mitochondrial branched‐chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82–85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched‐chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched‐chain amino acid catabolism is specific to each species. [ABSTRACT FROM AUTHOR]

A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich. L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352–360] and to be phosphorylated in vitro at serinc/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. d. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+ / calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate. [ABSTRACT FROM AUTHOR]

Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma;-carboxamide groups of protein-bound glutamine residues. These enzymes arc involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a eDNA of guinea-pig liver transglutaminase between the Ncol and Pstl sites of an expression vector, pKK233-2, and produced the Liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyt-terminal sequences. However, the α-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acctylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km, values for substrates in the histamine incorporation into acetyl α1¹-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the Nα-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

The brain specific S-100 protein is a mixture of two components S-100a and S-100 b with a subunit composition αβ or β2 respectively. The amino acid sequence of the β subunit has been previously determined. This paper presents the sequence of the α subunit in the S-100a protein. The α subunit consists of 93 amino-acid residues and has a relative molecular mass of 10400. The sequence shows extensive homology (58 %) with that of the β-subunit and shares an apparent calcium binding site in the C-terminal half of the molecule, suggesting a close evolutionary relationship between these subunits. [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]

The primary structure of the major parvalbumin (pI = 4.45) from the cartilaginous fish Raja clavata has been determined. The amino acid sequence was deduced by the analysis of peptides derived from tryptic digestion of the oxidized protein. These peptides were aligned by comparison with (a) overlapping peptides produced by limited tryptic and chymotryptic digestion, and (b) by comparison with the known structures of other fish parvalbumins. The molecular evolution of thornback ray parvalbumin is briefly discussed. [ABSTRACT FROM AUTHOR]

Sheep-serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch-gel electrophoresis, corresponding to a major and a minor fraction. This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM-cellulose and, finally (c) chromatography on DEAE-Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch-gel and polyamide-gel electrophoresis and by immuno-electrophoresis. The amino-acid compositions of both fractions were very similar and the sequences of the first eight amino-acid residues: Ser-Pro-Glu-Lys-Thr-Val-Arg-Trp-were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differences do not lie in the polypeptide chain. From the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N-acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic-acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low-speed equilibrium ultracentrifugation and by Archibald's method. The following physico-chemical constants have been established: absorption coefficient at 280nm, A1 mg/cm³1 cm = 1.25 ± 0.01 cm² · mg-1; partial specific volume, &vmacr; = 0.734 cm³ · g-1; sedimentation coefficient s20.wo = 5.15 S; refractive-index increment, dn/dc = 0.173 cm³ · g-1. [ABSTRACT FROM AUTHOR]

A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described. Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature. Yeast phosphoglyceromutase contains four highly similar, probably identical subunits of molecular weight 28000, a conclusion based on the following observations. Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge. Cyanogen bromide cleavage and tryptic digestion of the enzyme yield the number of peptides expected for identical subunits from the amino acid composition analysis. The purified phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate and 3-phosphoglycerate. It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3-bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction [Sasaki, R. et al. (1971) Biochim. Biophys. Acta, 227, 584–594, 595–607]. Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities. [ABSTRACT FROM AUTHOR]

The primary structure of protein S8 from the 30-S ribosomal subunit of Escherichia coil was determined mainly by automatic Edman degradation using a modified Beckman protein sequenator and the solid-phase sequenator of Laursen. The complete sequence, containing 109 amino acids, was derived by analysing peptides from tryptic, chymotryptic, thermolysin, staphylococcal protease and cyanogen bromide digestion of the protein. The amino acid composition was found to be (aspartic acid)6, (asparagine)3, (threonine)5, (serine)5, (glutamic acid)7, (glutamine)6, (proline)5, (glycine)6, (alanine)11, (valine)9, (methionine)4, (isoleucine)7, (leucine)9, (tyrosine)3, (phenylalanine)3, (lysine)11, (arginine)8, (cysteine)1. S8 is a basic protein and binds to the 16-S RNA; knowledge of its sequence is necessary for a detailed study of its interaction with the ribosomal RNA. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

The sequences of the 119 amino acids of bovine and porcine luteinizing hormone β-subunits were determined, 17 amino acid replacements being observed between these species. Variability in the primary structure was observed for porcine β-subunit, in that position 10 is occupied by both arginyl and glutamyl residues. No free amino terminal residue was detected for both porcine and bovine β-polypeptide chains and both exhibited carboxy-terminal heterogeneity. The homology of the β-subunits of luteinizing hormone from various species and the β-subunit of the bovine thyroid-stimulating hormone is discussed as the subunit of both hormones can combine with a similar α-subunit. [ABSTRACT FROM AUTHOR]

The primary structure of porcine luteinizing hormone α-subunit was established by determination of the amino acid sequence of its tryptic peptides, which were aligned by homology with the known sequence of ovine luteinizing hormone α-subunit. Its polypeptide chain is shorter by six amino acid residues at its amino-terminus, when compared to the ovine α-subunit. Furthermore four amino acid replacements are observed in the porcine as compared with the ovine chain. Heterogeneity of both carbohydrate prosthetic groups of porcine luteinizing hormone α-subunit is indicated by the different sugar compositions of the glycopeptides isolated in the course of this work. [ABSTRACT FROM AUTHOR]

The biosynthesis of melittin, the main toxin of bee venom, was studied in vivo by feeding radioactive amino acids to honey bees. Extracts from venom glands were analyzed for the presence of labeled melittin and other components. Radioactivity was first incorporated into another peptide which is considered to be a precursor of melittin. This conclusion is based on the observed labeling kinetics demonstrating the transient formation of this peptide. Furthermore, the structural similarity between melittin and this component could be established. Evidence is presented showing that it differs from melittin at the amino end. The precursor peptide could not be detected in the ejected venom. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

The present paper describes the effect of (+)-biotin on the metabolism of a freshly isolated strain of Desulphovibrio desulphuricans. In proliferating pure cultures of this Zürich strain on a synthetic substrate with ammonium chloride, lactate and sulphate an oxido-reduction process takes place according to the stoichiometric relation established by Baars (1930). In the medium mentioned (+)-biotin was shown to have favourable action when added in comparatively small amounts, whereas in presence of greater amounts of the vitamin a marked inhibition was observed. The maximum stimulation is induced at a concentration of 25 jig. of biotin per litre, while a pronounced inhibition sets in when the vitamin concentration is increased from 100 to 250 μg. per litre. It is proposed that the beneficial effect of biotin might be related to the action of the vitamin in the synthesis of aspartic acid and other amino acids from pyruvic acid, carbon dioxide and ammonia through Wood-Werkman condensation, amination and transamination. [ABSTRACT FROM AUTHOR]

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, ehymotrypsin and pepsin is reported. The data obtained permitted the assembly of a unique sequence of amino acids for the poly-peptide chain of the protein. Various corrections and one addition to the sequence previously communicated are made. [ABSTRACT FROM AUTHOR]

A protein is generally classified into one of the following four structural classes: all α, all β, α+β and α/β. In this paper, based on the weighting to the 20 constituent amino acids, a new method is proposed for predicting the structural class of a protein according to its amino acid composition. The 20 weighting parameters, which reflect the different properties of the 20 constituent amino acids, have been obtained from a training set of proteins through the linear-programming approach. The rate of correct prediction for a training set of proteins by means of the new method was 100%, whereas the highest rate of previous methods was 82.8 %. Furthermore, the results showed that the more numerous training proteins, the more effective the new method. [ABSTRACT FROM AUTHOR]

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Abstract: A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein–RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein–RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein–RNA interaction modeler). Given one or more protein data bank files of protein–RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein–RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein–RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/. [Copyright &y& Elsevier]

Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa toxin produced by certain pathogenic strains of Escherichia coli, constitutively activates members of the Rho GTPase family, leading to cytopathic effects. The toxin inhibits GTP turnover by Rho proteins through site-specific deamidation of a Rho glutamine required for GTP hydrolysis. To understand the basis for catalytic activity and target specificity of CNF1, the structure of a catalytically active fragment of CNF1 was sought. Here, strategies that led to successful expression of a soluble 33 kDa active fragment, growth and improvement in the quality of the crystals and determination of phases using a quadruple methionine-substitution mutant of the fragment are presented. [ABSTRACT FROM AUTHOR]

Polydocanol has a wide range of medical applications, especially in sclerotherapy of many diseases such as gastrointestinal antiplastia, oesophageal haemangioma etc. it is of interest to study the mode of binding of this medically important detergent and its subsequent action on proteins. Here, three crystal structures of serine protease trypsin are reported in the presence of varying concentrations of polydocanol in order to elucidate its mode of binding and interactions with proteins. Polydocanol binds to the protein with its hydrophilic head rather than the hydrophobic tail as is the case with other detergents such as SDS and MEGA-8. This hydrophilic binding mode results in the binding sites of polydocanol being distributed on the surface of the enzyme. There are at least 11 binding sites for polydocanol in trypsin. Polydocanol forms part of the large-scale water networks which connect distant regions of the enzyme, thereby stabilizing it. The hydrophilic binding of polydocanol also results in cross-linked pairs of tryp sin molecules. [ABSTRACT FROM AUTHOR]

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in the de novo biosynthesis of uridine 5'-monophosphate. In order to determine the structure of ODCase from Escherichia coli by the multi-wavelength anomalous dispersion technique, both native and SeMet-substituted proteins have been produced and purified. During the production of SeMet ODCase, it was observed that SeMet was the only amino acid that it was necessary to add to the defined medium during expression. SeMet-substituted ODCase in complex with the inhibitor 1-(5'-phospho-β-D-ribofuranosyl)barbituric acid crystallizes under similar conditions as the native enzyme. In contrast to the native enzyme, where the crystals belong to the orthorhombic space group P212121, the SeMet-substituted enzyme crystallizes in the monoclinic space group P21, with a quadrupling of the volume of the asymmetric unit. Despite the drastic difference in symmetry, the overall crystal packing is effectively identical in the two crystal forms. The change in space group appears to originate in differences in the crystal contacts near the SeMet and Met residues. These differences can be rationalized in terms of SeMet's larger size and hydrophobicity. [ABSTRACT FROM AUTHOR]

The structure of canavalin was refined to 2.1 and 2.0 Å resolution in cubic and hexagonal crystals of space group P213 and P63, respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary- structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the ioop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins. [ABSTRACT FROM AUTHOR]

Ionization of amino acids (AA) is very important concept in biochemistry. We integrate the mathematical concept of probability with biochemically relevant process of AA ionization. We visualize the ionization process with Mathematica software discussing intramolecular interactions between weakly acidic/basic functional groups and charge–pH variation of amino acids in water solution. The visualizations rely on the notion of probability of ionization of functional groups and demonstrate how the extent of ionization and charge varies with pH of the solution. The examples described include amino acids and weak diprotic acids and bases. The aim is to help students better appreciate the importance and consequences of AA ionization and correct some misconceptions. [ABSTRACT FROM AUTHOR]

In this paper we report proton two-dimensional NMR experiments on isolated α chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J-correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of α subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated α chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X-ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed. [ABSTRACT FROM AUTHOR]

In mouse spermatozoa, DNA is compacted by two protamines mP1 and mP2. Protamine mP2 (63 residues) is synthesized in spermatid nuclei as a precursor pmP2 (106 residues) which is subsequently processed at the end of spermiogenesis [Yelick, P. C., Balhorn, R., Johnson, P. A., Corzett, M., Mazrimas, J. A., Kleene, K. C. & Hecht, N. B. (1987) Mol. Cell. Biol. 7, 2173-2179]. Six proteins, three of which were described earlier [Chauvière, M., Martinage, A., Debarle, M., Alimi. E., Sautière, P. & Chevaillier, Ph. (1991) C. R. Acad. Sci. 313, 107-112], have molecular and electrophoretic properties similar to those of pmP2. They were isolated from purified testis nuclei and characterized by amino acid composition, N-terminal sequence and peptide mapping. From the amino acid compositions, it appears that all six proteins are rich in arginine, cysteine and histidine and are closely related to pmP2 and mP2. The N-terminal sequence of each protein overlaps a distinct region of the N-terminal part of pmP2. The C-terminal part of protamine mP2 starting at arginine 15 is common to all proteins as assessed by amino acid compositions and peptide maps, ll these structural data demonstrate that the six isolated proteins are products of pmP2 precursor processing. The six intermediate proteins pmP2/5, pmP2/11, pmP2/16, pmP2/20, pmP2/26 and pmP2/ 32 which contain 102, 96, 91, 87, 81 and 75 residues, respectively, are generated from the pmP2 precursor after N-terminal excision of 4, 10, 15, 19, 25 and 31 residues, respectively. The C-terminal sequence of protamine mP2 is strictly identical to that of its precursor; therefore, no maturation occurs in this part of the molecule. At the present time, the proteolytic pathway involved in the amino-terminal processing leading to the mature form of the protamine mP2 (63 residues) has not been elucidated. However, the different representation of six intermediates in the testis suggests that some stages of processing are faster than others or that some cleavage sites are preferred. The proteins described in this paper could result either from stepwise excision of N-terminal residues or from nonsequential cleavages. [ABSTRACT FROM AUTHOR]

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxoglutarate as a substrate of saccharopine dehydrogenase [ε-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming)] in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+M, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+M was shown to be the last reactant released. Saccharopine [ε-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E · NADH · saccharopine complex. [ABSTRACT FROM AUTHOR]

Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work [Airas, R. K. (1990) Eur. J. Biochem. 192, 401-409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg2+-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product. [ABSTRACT FROM AUTHOR]

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L- LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G→C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65°C but more stable above 70°C. [ABSTRACT FROM AUTHOR]

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PP1 exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine · ATP complex. Spermidine · ATP is a weaker substrate. The role of spermidine · ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines. [ABSTRACT FROM AUTHOR]

Interferon (α + β) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl cAMP. Core (2'-5')oligo(adenylate), i,e. A2'p5'A2'p5'A or (A2'p)2A, as welt as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2'p)2A of ornithine decarboxylase activity was reversed by dibutyryl cAMP. These results suggested that the activity of interferon was similar to that of (A2'p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity. [ABSTRACT FROM AUTHOR]

When Prototheca zopfii cells were pulse-labeled with 14C-containing amino adds and homogenized, 14C- labeled membranes were obtained. In vitro incubations with the previously labeled membranes and UDP-[³H]GIc produced a trichloroacetic-acid-insoluble fraction having both isotopes. A double-labeled glucoprotein was isolated and characterized. It has a relative molecular mass of 28000-30000 and a carbohydrate content of 10%. The oligosaccharide chain is linked to the protein through an O-glycosidic bond between hydroxyproline and glucose. The oligosaccharide has a polymerization degree ranging over 10-20 hexose units. Glucose is the only monosaccharide found; most of the glucose residues are β-l,4-1inked (90%) but some are β-l,3-1inked (10%). [ABSTRACT FROM AUTHOR]

High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed .The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high field methyl groups belonging to a valine residue lie directly over an aromatic ring. [ABSTRACT FROM AUTHOR]

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearotherrnophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: Multiple line equations cannot be represented in ASCII text. [ABSTRACT FROM AUTHOR]