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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.

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]

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.

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]

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]

From the venom glands of honey bees fed with different radioactive amino acids, labelled promelittin could be isolated. Extensive analysis of this precursor peptide has led to the conclusion that it contains the entire sequence of melittin, including the C-terminal glutamic acid diamide. At the amino end, promelittin was found to be heterogeneous. As compared to melittin, the main species contains eight additional amino acids. Other species of different chain length are present in varying amounts. The structure of the main component can be formulated as Glu-Pro-Glu-Pro-Asp-Pro-Glu-Ala-melittin(1–26). The methodology used for determining the sequence of radioactive peptides is discussed. [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]

In the previous paper in this series it was shown that a pure flavin pentapeptide isolated from hepatic monoamine oxidase contains I mole each of serine and tyrosine and 2 moles of glycine, as well as an additional amino acid which is covalently linked to the 8α carbon of riboflavin. This amino acid has been identified as cysteine and the linkage to the flavin as a thioether on the basis of positive chloroplatinic and negative iodine-azide tests, performic acid oxidation or reduction with zinc, followed by acid hydrolysis, which yield cysteic acid or cysteine, respectively. The fluorescence properties of the flavin from monoamine oxidase agree with those expected for a thioether and its oxidation products. In regard to optical and electron spin resonance spectra, chemical stability, and fluorescence characteristics the flavin peptide isolated from the enzyme agrees excellently with the properties of synthetic cysteinyl 8α-riboflavin. [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]

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]

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]

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]

Tryptic and peptic peptides of the L-4 light chain of chicken skeletal muscle myosin were isolated. The amino acid sequences of all the tryptic peptides were determined. The alignment of the tryptic peptides in the protein was deduced from their homology with the primary structure of the A2 (L-4) light chain of rabbit skeletal muscle myosin. The compositions of the peptic peptides confirmed the alignment. Comparing the whole sequence of the L-4 light chain thus established with that of the A2 light chain of rabbit skeletal muscle myosin, 24 amino acid substitutions, were recognized. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of 4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized. [ABSTRACT FROM AUTHOR]

The primary structure of the calf thymus non-histone chromosomal protein HMG-17 has been determined. The sequence was determined mainly from data provided by the peptides obtained by cleavage with staphylococcal protease. Additional information was obtained from peptides produced by cleavage with trypsin and α-protease (from Crotalus atrox venom) and by partial acid hydrolysis. The protein is 89 amino acid residues in length, and has molecular weight of 9247. The N-terminal two-thirds of the molecule is highly basic, 22 of the first 58 residues being lysine or arginine, whereas only seven residues are aspartic or glutamic acid residues. In contrast, the C-terminal region of the molecule has an overall negative charge, only four of the last 31 residues being basic, whereas seven aspartic and glutamic acid residues are present. The protein is also characterised by a region of high density of proline residues, there being six proline residues between residues 31 and 40. A region of 19 residues sequence similarity with the trout-specific histone, H6, is noted together with some smaller regions of sequence similarity with histones H1 and H5. [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]

Three different glycoasparagines have been isolated from the urine of a patient with aspartylglycosaminuria and their structures determined using sugar, amino acid, and methylation analysis, enzymic degradation and measurements of the optical rotations. The structures were 2-acetamido-1-N-4'-L-aspartyl)-2-deoxy-β-D-glucopyranosylamine(yield 135 mg/l) β-D-galactopyranosyl-(1→4)-2-acetamido-1-N-(4'-L-aspartyl)-2-deoxy-β-D-glucopyranosylamine (yield 35 mg/l), and α-D-mannopyranosyl-(1 → 6)-beta;-D-mannopyranosyl-(1→4)-acetamido-2-deoxy-β-D-glucopyranosyl-(1 → 4)-2-acetamido-1-N-(4'-L-aspartyl)-2-deoxy-beta;-D-glucoyranosylamine (yield 30 mg/l). The first two compounds have previously been described, whereas the third compound is different from any of the glycoasparagines isolated before. [ABSTRACT FROM AUTHOR]

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin. [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]