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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

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 several research papers related to biochemistry that would be published in coming issues of the European Journal of Biochemistry. Some of the research topics include: differentiation of ovine brown adipocyte precursor cells in a chemically defined serum-free medium; production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae; binding of sugar phosphates, inositol phosphates and phosphorylated amino acids to actin; etc.

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.

A new approach of predicting structural classes of protein domain sequences is presented in this paper. Besides the amino acid composition, the composition of several dipeptides, tripeptides, tetrapeptides, pentapeptides and hexapeptides are taken into account based on the stepwise discriminant analysis. The result of jackknife test shows that this new approach can lead to higher predictive sensitivity and specificity for reduced sequence similarity datasets. Considering the dataset PDB40-B constructed by Brenner and colleagues, 75.2% protein domain sequences are correctly assigned in the jackknife test for the four structural classes: all-α, all-β, α/β and α + β, which is improved by 19.4% in jackknife test and 25.5% in resubstitution test, in contrast with the component-coupled algorithm using amino acid composition alone (AAC approach) for the same dataset. In the cross-validation test with dataset PDB40-J constructed by Park and colleagues, more than 80% predictive accuracy is obtained. Furthermore, for the dataset constructed by Chou and Maggiona, the accuracy of 100% and 99.7% can be easily achieved, respectively, in the resubstitution test and in the jackknife test merely taking the composition of dipeptides into account. Therefore, this new method provides an effective tool to extract valuable information from protein sequences, which can be used for the systematic analysis of small or medium size protein sequences. The computer programs used in this paper are available on request. [ABSTRACT FROM AUTHOR]

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried salt- bridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family. [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]

Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambigous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains sites 1 and 2; TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of sites 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labelled peptides may be completely separated and quantified by high-voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and-formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to >95 % accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5% and 90%) >98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was <2 % if at all. Relative initial rates of phosphorylation, site 1:site 2:site 3 were approximately 90:3:1. [ABSTRACT FROM AUTHOR]

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]

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences near the peroxidase heme prostetic group, in the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heine, and 34 residues around the probably distally located histidine, and compare them with the histidine-containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3 and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physico-chemical and enzymic properties. Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, P2 and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site. [ABSTRACT FROM AUTHOR]

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion- exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmote into four by paper charmatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde. [ABSTRACT FROM AUTHOR]

1. The purification of a halidohydrolase from cell-free extracts of Pseudomonas dehalogenans NCIB 9061 grown on glucose and monochloroacetate as carbon source, is described. 2. This enzyme catalyses the hydrolysis of some 2-halogen substituents on aliphatic carboxylic acids, releasing a halide ion and a hydrogen ion and causing inversion of the configuration. 3. The molecular weight as determined by chromatography on Sephadex G-100 was about 15000. 4. The optimum activity was at pH 9.4. Activity was rapidly and irreversibly lost below PH6.0. 5. The amino acid composition of the purified enzyme in its native state and after photooxidation in the presence of methylene blue is presented. 6. The variation of the kinetic parameters, V and Km between pH 7.0 and pH 10.0 suggest that a group with a pKa of about 7.8 is involved in its base form in the catalytic reaction. 7. The enzyme is rapidly inactivated by reaction with iodine and upon photooxidation in the presence of methylene blue, but the effect of thiol reagents suggests that a thiol group is not directly responsible for catalysis. 8. It is suggested that a histidine residue is implicated in the catalytic reaction and a mechanism is proposed which is consistent with the experimental results. [ABSTRACT FROM AUTHOR]

In paper [5] a simplified model for the hydrophobic binding sites of trypsin was proposed, showing where 1-amino-n-alkanes and α,ω-diamino-n-alkanes are bound while they competitively inhibit the catalyzed hydrolysis of a substrate ester. In the model, the enzyme bas two hydrophobic binding sites separated by an aqueous region which presumably contains the catalytic important residues. The first site binds up to 4 methylene groups of the aliphatic side chain adjacent to the ω-amino group: additional—CH2 groups in t, he chain extend into the aqueous region. If the chain is larger than 7 carbon atoms, the additional methylene or methyl groups are bound to the second hydrophobic site. This site is found to bind less strongly than the first site. This paper presents a thermodynamic investigation of the binding properties of trypsin in the formation of complexes with cationic esters and amids which bear an ammonium group on either the end of the acid side chain or the end of the alcohol or amine component. Free energy and in addition enthalpy and entropy of binding were measured as a function of the length of the inhibitor molecule and as a function of the position of the ester or amide carbonyl group. It was found that thc stability of a complex increases linearly with length of the molecule, and that the magnitude of increase depends on the position of the carbonyl group. Amides always exhibit a smaller increase than esters. Inhibitors with the ammonium group on the alcohol component, were bound with about, the same stability than the corresponding ω-aminoacidesters. The results are consistent, with the model. Positioning of the carbonyl group within the first or second binding site reduces stability due to restricted interaction of the hydrophobic residues on enzyme and inhibitor. Only when the carbonyl group falls into the aqueous region, the free energy for an inhibitor containing the ester group is similar to that for a 1-amino-n-alkane with identical chain length. Enthalpy and entropy as functions of the position of the carbonyl group show minima for the position within the aqueous region. This observation is correlated with the jump of enthalpy and entropy of complex formation of trypsin in the series of 1-amino-n-alkanes reported earlier. It is shown that the magnitude of free energy of complex formation with specific substrates is well estimated on basis of the model. As a consequence, the alternative binding of either the 2-N-acyl group or the alcohol or amine component, to the second hydrophobic site may imply a mode of less productive binding. It is emphasized that unproductive binding should be reflected in the Michaelis Menten constants for substrate amides whereas thc Michaelis Menten constant, for a substrate ester is normally not perturbed. [ABSTRACT FROM AUTHOR]

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radio- active malonyl and methyhnalonyl enzymes are formed and may be precipitated with trichloro- acetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages. Methyl- [3-14C]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methyl)malonyl peptides. were separated into three different fractions A, B and C. In peptides B and C (methyl)malonic acid is bound by performic acid stable linkages, whereas (methyl)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor sateffite of peptide B, the quantitative relation of both varying somewhat in different preparations. it is concluded that both are derived from a common original peptide sequence in the enzyme. Methyl- [3-14C]malonyl peptide B was further purified by the following procedures: Dowex-50X2 chromatography, subtilisin S digestion, DEAB-Sephadex chromatography, Dowex-50X2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B1and B2, resulted from subtilisin S treatment. They finally were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: B1 = Leu,AIa,Gly,Ser,His and B2 = Leu, Ala,Gly(2 × ),Glu,Ser,His, Leucine was the C-terminal amino acid in peptide B1. No cysteine was found in either peptide. A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAE- Sephadex, Dowex-50X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl B1peptide B2. The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methyl)malonic acid in peptides B and C is bound to serine by an O-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine, [3-14C]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, β-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phospho- pantetheine. 4'- Phospho-pantetheine, therefore, represents the so called "central" SR-group in yeast fatty acid synthetase, The other malonyl binding site is thought to be the active center of the malonyl transferase in the complex. After alkali treatment of the purified fatty acid synthetase under defined conditions 4' -phospho - pantetheine was isolated from the bydrolysate and characterized as S-benzoyl-4'-phospho- pantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules of 4'-phospho-pantetheine. According to the amount of malonyl enzyme formed, not all of the enzyme-bound phospho-pantetheine residues accept malonic acid. Some may be involved in other functions. [ABSTRACT FROM AUTHOR]

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αs1-casein, a single-chain phosphoprotein, formerly reposed to have arginine as an NH2-termimal, and Leu-TrpOH as COOH-terminal residues. ryptic digestion of maleyl αs1-casein (genetic variant B), in theory bruited to arginyl bonds, yields eight peptidic fragments, Tm1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The ammo acid composition and the phosphate content of these fragments were determined. The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. Thus, six fragments only—2, 4, 5, 6 and 7—arise from the specific cleavage of the αs1-casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2-terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to rcpresent respectively the NH2- and COOH-terminal parts of the αs1-casein chain. These peptidic fragments—except Tm4 which is devoid of lysine—were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established. As a control, the tryptic peptides of αs1-casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride. In summary, this first step of our work gives: (a) the composition of ail the bovine αs1-casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain: (c) confirmation that the molecular weight of the bovine αs1-casein monomer is approximately 23600. [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]

In a previous paper, we reported on the structural properties of a 35-residue peptide corresponding to a modified basic subdomain (bSD) of the basic zipper protein c-Jun (residues 252–281) as determined by combined use of 1H-NMR, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopies [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. (1995) Eur. J. Biochem. 231, 370–380]. The fragments NP and CP (the N-terminal residues 1–19 and C-terminal residues 16–35 of bSD, respectively) proved to be particularly useful for the assignment of the 1H-NMR spectra of the full-length bSD, which has been achieved completely in aqueous solution and partially in trifluoroethanol. Here, we report on the structural properties of NP and CP in aqueous solution and under varying H2O/trifluoroethanol conditions, again using 1H-NMR, CD and FT-IR experiments. Both CD and FT-IR results established that the fragments are weakly structured in aqueous solution. Addition of trifluoroethanol to aqueous solutions of the peptides produced their stabilization into helix, following a profile sigmoidal for NP and nearly linear for CP. Quantitative NOEs, secondary · Hα chemical shifts, NH temperature coefficients and 3JαN coupling constants for the peptides in aqueous solutions provided indications for weak helix features (nascent helices) manifested within two sites (continuous dNN NOEs) in both NP and CP. For each peptide, an excellent agreement was observed between experiments and predictions with the AGADIR program for the location of these nascent helices in the sequences. Trifluoroethanol provoked both the α-helix stabilization within these sites and the α-helix propagation to adjacent amino acid residues. Finally, our results reflected the high flexibility and helix potential of the NP and CP fragments, these two properties seeming crucial for the accomodation of c-Jun to its specific DNA targets. The results demonstrated also the fragmentation's benefits in dissecting a protein or a complex peptide into smaller fragments and analyzing their structure individually. [ABSTRACT FROM AUTHOR]

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The confirmation of the syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three dimentional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported. [ABSTRACT FROM AUTHOR]

Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. [ABSTRACT FROM AUTHOR]

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide · HCI (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat, values of these chimeras. From the data obtained, two sequences, i.e. 62–103 and 146–220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2-- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other. [ABSTRACT FROM AUTHOR]

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). [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]

Preparations of bovine lung surfactant D (SP-D) and conglutinin were examined by electron microscopy, gel-filtration and SDS/PAGE. SP-D is composed of non-covalently linked subunits, of 160 kDa, which each contain three, disulphide-linked, 44-kDa polypeptide chains. In the electron microscope a single 160-kDa subunit of SP-D appears as a 45.8 ± 3-nm-long rod connected to a small globular ‘head’. Particles were also seen which correspond to non-covalently linked dimmers, trimers and tetramers of the 160-kDa monomer subunit of SP-D. The tetramer structure contains 12 polypeptide chains and is very similar to the electron microscopy images and model reported by Strang et al. [Strang, C. J., Slayter, US., Lachmann, P. J. and Davis, A. E. (1986) Biochem. J. 236, 3811–389] for bovine conglutinin in which four 160-kDa subunits are disulphide-linked to give a molecule of expected molecular mass of 528 kDa. This study confirmed the findings by Strang et al. in the above paper for intact conglutinin and also emphasized that the rod-like structures, of length 37.6 ± 3.7 nm. Seen in the conglutinin subunits were significantly shorter than those in SP-D despite the close similarity in amino acid sequence (79% identity) and chain length between the two proteins. In addition, a truncated form of conglutinin was found in the conglutinin preparations, due to limited proteolysis of the Arg-Ala bond at position 54 in the 44-kDa chains. These truncated conglutinin chains yield a subunit composed of three shortened, non-disulophide-linked, chains and this subunit appears as a monomer with a rod length of 34.2 ± 2.8 nm in the electron microscope. On gel-filtration, a proportion of the SP-D preparation behaved, as expected, as a molecule with an apparent molecular mass of 600 kDa. The remainder of the SP-D preparation behaved as aggregated material with a molecular mass greater than 900 kDa which yielded no distinct structures in the electron microscope. Intact conglutinin was wluted at a position greater than 900 kDa but yet provided clear electron microscopy images of the tetramer structure described above. Although it is difficult to explain fully the anomalous behaviour of SP-D and conglutinin on gel-filtration, it is proposed that the natural form of both Sp-D and conglutinin is the highest distinct oligomer form seen in the electron microscope, i.e. a tetramer of the 160-kDa subunits in which the four rod-like elements are linked in a tail-to-tail fashion to a central core. [ABSTRACT FROM AUTHOR]

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

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]

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]

Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, I1e85 δ methyl and Ile9 γ methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed. [ABSTRACT FROM AUTHOR]

Site-specific mutagenesis has been used to replace amino acid residues in the active site of human carbonic anhydrase II with residues characterizing carbonic anhydrases I. Previous studies of [Thr200→ His]isoenzyme II [Behravan, G., Jonsson, B.-H. & Lindskog, S. (1990) Eur. J. Biochem. 190, 351–357] showed that His200 is important for the specific catalytic properties of isoenzymes I. In this paper some properties of two single mutants, Asn62 → Val and Asn67 → His, as well as a double mutant, Asn67 → His/Thr200 → His, are described. The results show that neither Va162 nor His67 give rise to isoenzyme-I-like properties, while the double mutant behaves like the single mutant with His200. At pH 8.9, the variant with Va162 has a higher value of kcat/Km for CO2 hydration than unmodified isoenzyme II, whereas the variant with His67 has an enhanced kcat value. The replacement of Asn62 with Val results in a 20% increase of the 4-nitrophenyl acetate hydrolase activity. For the double mutant, the esterase activity is quite close to that calculated on the assumption that the effects of the two single mutations on the free energy of activation are additive. [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]

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [³H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/ or long-term labelling with [35S]sulfate and [³H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins. [ABSTRACT FROM AUTHOR]

The paper describes further characterization of the 55-kDa short-chain collagen from lens capsule. Lens capsules were extracted with 5.5 M guanidine · HCl and the extracted material was fractionated on agarose A5M followed by high-pressure liquid chromatography (HPLC). By amino acid composition, the major fraction obtained from HPLC was found to be different than type-IV collagen fragments. The 55-kDa short-chain collagen on pepsin digestion produced a 45-kDa pepsin-resistant fragment. The undifferentiated embryonal carcinoma (F-9) cells were found to synthesize increased amounts of 55-kDa short-chain collagen. The identity of this biosynthesized molecule with 55-kDa short-chain collagen from lens capsules was established by immunoprecipitation experiments. The results indicated a close similarity or identical nature of the short-chain collagens from these two sources. [ABSTRACT FROM AUTHOR]

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

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

Bacterially synthesized, recombinant-DNA-derived bovine growth hormone (r-bGH), prepared as described in the preceding paper in this journal, has been characterized in comparison with pituitary bovine growth hormone (pit-bGH). The characterization criteria include sodium dodecyl sulfate/polyacrylamide gel electrophoresis, automated N-terminal sequence analysis, amino acid composition, isoelectric focusing, reverse-phase high- performance liquid chromatography, ultraviolet absorbance, analysis for free protein thiol, sizing by gel filtration, circular dichroism, radioimmunoassay and biological activity in the hypophysectomized rat weight-gain assay. In every respect the r-bGH appears to be virtually identical to pit-bGH. [ABSTRACT FROM AUTHOR]

An 11450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptornyces R61 was cloned in Streptornyces lividans using the high-copy-number plasmid pi J702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DDpeptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine β-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coil ampC β-lactamase (class C). Since the Streptomyces R61 DD-peptidase and β-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the β-1actamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the β-lactamases of classes A and C are related in an evolutionary sense. [ABSTRACT FROM AUTHOR]

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II ts. Although the apparent affinity of tRNA for site U remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed. [ABSTRACT FROM AUTHOR]

An inhibitor for S-adcnosyl-L-methionine (AdoMet)-dependent metbyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two- dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyitransferases examined. [ABSTRACT FROM AUTHOR]

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the sucking mouse assay respectively. [ABSTRACT FROM AUTHOR]

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-biphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phophofructor-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation. [ABSTRACT FROM AUTHOR]

According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made. [ABSTRACT FROM AUTHOR]

The partial ammo acid sequence of dihydrofolate reductase (DHFR. EC 1.5,1.3) from human KB/6b cells hi been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradation and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequence. A comparison of the proposed human DHFR sequence with the previously known sequent of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Owls and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704. 163- 173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight micrasornal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine- 15 by arginine. This paper brings the final proof that two iso-cytochromes b5- exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed. [ABSTRACT FROM AUTHOR]

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B, Reich, E., and Tatum. E. L. (1963) Proc. Natl Acad. Sci. USA, 50. 175–181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics. [ABSTRACT FROM AUTHOR]

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37 % identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the half- molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransfernn is compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached. [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]