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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]

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]

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]

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]

The complete amino acid sequence of turnip peroxidase TP 7, the principal isoperoxidase during winter in turnip, Brassica napus L., variety Blanc dur d'hiver, has been determined by sequence analysis of cyanogen bromide fragments and of tryptic peptides. The turnip peroxidase TP 7 enzyme is composed of 296 amino acids, one hemin group and one neutral carbohydrate side chain attached through asparagine. The molecular weight of the polypeptide part is 31 060, and including heroin and carbohydrate the molecular weight of the native enzyme is close to 33 400. The isoelectric point of turnip peroxidase TP 7 is 11.6. Comparison of turnip peroxidase TP 7 and horseradish peroxidase HRP C shows that they contain four similarly located disulfide bridges and have pyrrolidone carboxylyl N termini. Their common evolutionary origin is distant as their amino acid sequences are only 49% identical. Furthermore, turnip peroxidase TP 7 differs significantly from three other isoperoxidases of turnip root, turnip peroxidases TP 1, TP 2 and TP 3, and from horseradish peroxidase HRP C in its physico-chemical and enzymatic properties, and its pronounced season-dependent appearance. All these differences of turnip peroxidase TP 7 and of the others suggest they serve separate biological functions. [ABSTRACT FROM AUTHOR]

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

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

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

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD-peptidase was fragmented by (a) cyanogen bromide cleavage of the carboxymethylated protein, (b) trypsin digestion of the carboxymethylated protein and (c) trypsin digestion of the protein treated with β-iodopenicillinate and endoxo-Δ14-tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, β-iodopenicillanate reacted selectively with the active site of the DD-peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active-site peptide. [ABSTRACT FROM AUTHOR]

1. The amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus has been elucidated. 2. The polypeptide contains 332 amino acids and its sequence is 70% identical with that of the enzyme from the moderate thermophile Bacillus stearothermophilus. 3. In contrast to less thermostable forms of the enzymes from B. stearothermophilus, pig, lobster and yeast, the T. aquaticus enzyme has only one cysteine residue, namely cysteine-149 which is required for catalysis. [ABSTRACT FROM AUTHOR]

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at position 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions. [ABSTRACT FROM AUTHOR]

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

Bence-Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion-exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion-exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic pepfides were isolated and characterized to obtain overlapping peptides for the whole V-region and for some tryptic peptides of the C-region. Protein DEL is related to subgroups III and IV of the γ-chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups IH and IV but in contrast to subgroups I and II of the γ-chains. However, marked differences exist between protein DEL and the proteins of subgroups IH and IV. a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL. b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the γ-chains. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pk of the group to be dansylated. A complete chromatographie separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidiy. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N-terminal amino acids, and 5-10 nmoles for their quantitative estimation. Some examples (α-chymotrypsin, trypsinogen, ribonuclease, lysozyme aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method. [ABSTRACT FROM AUTHOR]

A new procedure for the determination of the N-terminal groups in peptides and proteins is described. The free amino-groups are allowed to react with 2-fluoro-3-nitropyridine or 2-fluoro- 5-nitropyridine in slightly alkaline solution, the resulting derivatives are easily hydrolyzed in dilute hydrochloric acid in sealed tubes to give the corresponding nitropyridyl amino acids. Most of these compounds are quantitatively recovered under these conditions; in particular it is possible to estimate proline and glycine as N-terminal residues. After isolation the nitropyridyl amino acids can be estimated spectrophotometrically; in some cases it is possible to regenerate the parent amino acid by treating the derivatives with strongly alkaline solutions. The adequacy of the present techniques in relation to those of other workers was tested on insulin, lysozyme and aldolase; the advantages and peculiarities in comparison with these procedures are discussed. [ABSTRACT FROM AUTHOR]

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

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin–peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines. [ABSTRACT FROM AUTHOR]

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

We have studied the structures of synthetic peptides which correspond to the proposed first and second membrane-spanning segments of the human red cell anion transporter (band 3). The peptides, which were acetylated at their N-termini and amidated at the C-termini, comprise the 20 amino acids of residues 405-424 and 21 amino acids of residues 436-456 of the human band 3 sequence. The solution structures of the peptides in trifluoroethanol were studied by two-dimensional NMR spectroscopy. Characteristic NOEs were observed indicating that the peptides adopted a predominantly α-helical structure in trifluoroethanol solution. Dynamical simulated annealing using the program XPLOR was employed for the structure calculations. The amide exchange rates in trifluoroethanol have also been measured and are consistent with an α-helical structure for the peptides. [ABSTRACT FROM AUTHOR]

A protein-O-D-mannosyltransferase (PMT) assay was optimized using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac YNPTSV-NH2 was resistant to proteolysis and was apparently a better acceptor for o-mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a km of 0.075 nM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH2 whilst no incorporation was observed with AC-GTG-NH2. [ABSTRACT FROM AUTHOR]

The local specificity determinants for a T-cell protein tyrosine phosphatase (TC-PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T-cell PTP, deprived of its C-terminal down-regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C-terminal down-regulatory site of pp60c-src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c-src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal-growth-factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to I μM concentration. The replacement of either Ser-P or O-methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src-(41 2-418), src-(414 -418) and abl-(390 -397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N-terminal side of phosphotyrosine, with special reference to position -3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur. By replacing the truncated form of TC-PTP with the full length TC-PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C-terminal regulatory domain of TC-PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets. [ABSTRACT FROM AUTHOR]

A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant Cterminal region (from residues 14-27), including an amidated carboxyl end and a variable N-terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182-189]. By using synthetic oligonucleotides corresponding to the C-terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombininlike peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli. [ABSTRACT FROM AUTHOR]

The sequence of very basic proteins such as protamines (more than 50% arginines) and related peptides has been determined using mass spectrometry in conjunction with Edman degradation. The capabilities of three mass spectrometric (MS) techniques [fast-atom-bombardment (FAB), 252Cf plasma desorption (252CFPD) and electrospray (ES)] have been evaluated on stallion protamine 1, cuttlefish protamine, and the corresponding cleavage peptides. In contrast to FAB-MS and 252Cf PD-MS. ES-MS made possible an easy determination of the molecular mass of the intact protamines (≈8 kDa). With ES-MS about 0.2 nmol was sufficient to yield a mass measurement with an accuracy of 0.05%. On peptides smaller than 3500 Da, both FAB-MS and 252Cf PD-MS allowed mass measurements with an accuracy of 0.1%. 252Cf PD-MS appeared more sensitive than FAB-MS by about a factor of 10. FAB-MS is nevertheless particularly interesting since in most cases it produced spectra with intense A-type fragmentation ions which provided reliable primary structure information. [ABSTRACT FROM AUTHOR]

Energetically favored conformations of glycopeptide I were calculated using the newly developed force-field program. GEGOP (geometry of glycopeptides). The three-dimensional structure of glycopeptide 1, which is part of the Fc fragment of IgG1, has been calculated. I contains 27 amino acid residues from Pro291 to Lys317 and a biantennary decasaccharide N-linked to Ash297. The conformations of the peptide and the carbohydrate parts are shown to be mutually dependent. Single glycosyl residues of I exhibit interaction energies of up to -31.8 kJ/mol with the peptide portion. Generally, only a few of the glycosyl residues of the oligosaccharide moiety express significant interaction energies with the peptide parts. No easy prediction is possible of glycosyl residues which exhibit favorable interaction energies. However, in all of the calculated structures, the glycosyl residues of the 1 - 6-linked branches show strong attractive forces for the peptide part. 1 - 6-glycosidically linked branches can adopt a larger number of conformations than other linkages due to their high flexibility which allows more favorable interactions with proteins. We developed the GEGOP program in order to be able to study the preferred conformations of large glycopeptides. The program is based on the GSA (geometry of saccharides) program and utilizes the HSEA (hard sphere exo anomeric) force field for the carbohydrate cart and the ECEPP/2 (empirical conformation energy program for peptides} force field [Némethy. G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 87, 1883 1887] for the peptide part. The GEGOP program allows the simultaneous relaxation of all rotational degrees of freedom of these glycoconjugates during the energy optimization process. Thus. mutual interactions between glycosyl and amino acid residues can be studied in detail. [ABSTRACT FROM AUTHOR]

The sequence-specific assignment of resonances in the 500-MHz ¹H-NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin-A, is described. The assignment procedure involved analysis of two-dimensional phase-sensitive multiple-quantum-filtered, double-quantum, homonuclear Hartmann-Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues Resonances arising From a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Goolcy, P. R. and Norton, R. S. (1985) Fur. J. Biochem. 153, 529–539] and provide the basis for a more accurate definition of the conformation of anthopleurin-A in aqueous solution. The secondary structure includes a four-stranded antiparallel β-sheet encompassing residues 2–4, 21–2, 34–36 and 45–49, and possibly a β-bulge located at Ser-19 and Gly-20. A type II β-turn is formed by residues 30–33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro-41 appears to be unique to anthopleurin-A. [ABSTRACT FROM AUTHOR]

1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the eDNA sequence [López-Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85. 5784–5788] shows that this site corresponds to Serl215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Serl200. A total of eight serine residues phosphorylated in, vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Serl215) are located in the central region. 3. Prior phosphorylation of Ser77 and Serl200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Set79 and Serl200, but not Serl215. by the AMP-activated protein kinase. Phosphorylation of Serl215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Serl200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Serl200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino- terminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Serl200 and Serl215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77. [ABSTRACT FROM AUTHOR]

The S′-subsite specificity of bovine pancreatic α-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S′ binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude. [ABSTRACT FROM AUTHOR]

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 μg) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict tour N-linked glycosylation sites in the P40 molecule. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of winged bean albumin-1 (WBA-1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA-1 was found to be homologous with the Kunitz-type seed trypsin inhibitors. The similarity between WBA-I and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C-terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz-type proteins and the carboxy-terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of β sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of β sheet to that found for soybean trypsin inhibitor by X-ray diffraction studies. [ABSTRACT FROM AUTHOR]

Rat-liver biliverdin reductase exists in two molecular forms. The major form I has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form I was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD +-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore saught. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDg/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme. [ABSTRACT FROM AUTHOR]

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1. [ABSTRACT FROM AUTHOR]

The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a. Ki = 0.4 mM. Binding of [³H]carboxyatractylate and [³H]bongkrekate is largely inhibited by pyridoxal-P treatment with K1 approximately 1 mM. [³H]Carboxyatractylate is not and [³H]bongkrekate weakly removed by pyridoxal-f, whereas [³H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [³H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [³H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysinecontaining peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP ⇄ MgADP + ADP) from baker's yeast has been determined. 1. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid-phase sequencer or a gas-phase sequencer. 2. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys-C and cyanogen bromide cleavages. 3. The N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. 4. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages. 5. The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active-site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP : AMP phosphotransferase from beef heart mitochondria. [ABSTRACT FROM AUTHOR]

The vitelline coat lysin of a top shell, Tegula pfeifferi, is a single polypeptide consisting of 118 amino acid residues and having a relative molecular mass of 13800. The complete amino acid sequence of the vitelline coat lysin was determined by the analyses of five peptides obtained by cyanogen bromide degradation and three fragments obtained by Staphylococcus aureus protease digestion of the protein. The sequence showed the presence of microheterogeneities in the vicinity of the C-terminal half of the molecule and the existence of two homologous domain structures.

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF, rEGF competes for the binding of 125l-labelled mEGF to human carcinoma cells with the same affinity as mEGF, rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2–48), rEGF(3–48) and rEGF(4–48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 μg protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine. phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn- Ser- Asn- Thr- Gly- Cys- Pro- Pro- Ser- Tyr- Asp- Gly- Tyr- Cys- Leu- Asn- Gly- Gly- Val- Cys- Met- Tyr- Val- Glu- Ser- Val- Asp- Arg- Tyr- Val- Cys- Asn- Cys- Val- Ile- Gly- Tyr- Ile- Gly- Glu- Arg- Cys- Gln- His- Arg- Asp- Leu- Arg. The calculated relative molecular mass from the sequence analysis is 5377. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence (72 amino acid residues) of a double-headed proteinase inhibitor from seeds of Vicia angustifolia L. var. segetalis Koch has been determined and compared with those of other double-headed inhibitors of known structure. Sequencing was performed by conventional methods with the aid of the fragments produced by reduction and S-carboxymethylation of the enzymatically modified inhibitors, and also using tryptic and chymotryptic peptides. The positions of the 14 half-cystine residues agreed among all the reported primary structures of the legume double-headed inhibitors. However, V. angustifolia inhibitor possessed extensive amino acid differences compared to the others. The phylogenetic relationship among these inhibitors was established using the unweighted pair-group method and revealed that the V. angustifolia inhibitor and the peanut inhibitor B-III had diverged at a relatively earlier stage compared to the other inhibitors. [ABSTRACT FROM AUTHOR]

The primary structure of the most basic (pI = 4.88) of the two major parvalbumins from frog skeletal muscle (Rana esculenta) has been determined by partial automatic sequencing of the protein which exhibits a free N terminus, and a study of overlapping peptides obtained by cyanogen bromide cleavage and digestion with trypsin, thermolysin and Armillaria mellea protease. This protein shows the typical structure of an α-parvalbumin. Comparison of the primary structure of ion binding loops of α and β-parvalbumins does not provide a clear-cut explanation of their differences in ion-binding properties. [ABSTRACT FROM AUTHOR]

By comparison with what is known of disaccharides transport, it has been suggested that intestinal aminopeptidase N could, hydrolyze, on the surface of the microvillus membrane, oligopeptides longer than tripeptides and itself subserve the translocation function for the amino acids released from these peptides. This article describes the synthesis of the tritiated azido-tetrapeptides p-azido[³H]phenylalanyl-alanyl-glycyl-glycine containing L or D-alanine. The synthesized products possess a function which displays all the characteristics of an aryl-azide. The photosensitive tetrapeptide formed with Lala-Gly-Gly is as good a substrate for porcine and rat aminopeptidases N as unmodified peptides while the tetrapeptide formed with DAla-Gly-Gly is not hydrolyzed at all. In addition a pattern of stepwise hydrolysis could be demonstrated and aminopeptidase N is the only exopeptidase present in the mucosal cells capable of utilizing the modified tetrapeptide as substrate. Uptake assays performed on everted rings of rat jejunum with the azido-tetrapeptide as substrate have shown that: (a) the azido-tetrapeptide is not transported intact but must be hydrolyzed first; (b) p-azido-phenylalanine is not released in the external medium and therefore its observed uptake is not from the bulk medium and (c) the azido-D-tetrapeptide is only accumulated by passive diffusion. These observations suggest the presence on the brush border membrane of an aminopeptidase-related transport system. [ABSTRACT FROM AUTHOR]

The tripeptide Nα,N∊-diacetyl-lysyl-D-alanyl-D-alanine (Ac2-LLys1-DAla2-DAla3), which is the standard substrate of the Zn2+G and serine R61 D-alanyl-walanine peptidases, and several LDD tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short-range and long-range interactions. Among the many backbone conformers of minimal energy of the &phis;i,ψi space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90 % or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ΣP values arc still as high as 35-50 %. All the above tripeptides bind to the serine D-alanyl-D-alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+ D-alanyl-D-alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1. 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2-LLys-DAla-DAla show that the main backbone has a tendency to adopt a ring-like shape from which the LLys side chain protrudes as an extended structure. This latter structure forms with the C-terminal D-alanyl-D-alanine an angle varying between 120° and 180° (depending on the conformers) so that its N-terminal acetyl group is about 1 -1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a DAla at position 2 with both D-alanyl-D-alanine peptidases and at position 3 in the case of the serine D-alanyl-D-alanine peptidase. Finally, all the conformers of the LAla2 and LA1a3 analogues of Ac2-LLys-DAla-DAla fall outside the backbone conformational space that comprises the &phis;i&phe;i angles exhibited by the four types of conformers of the LDD tripeptides. The LAla2 and LAla3 tripeptide analogues do not bind to the serine D-alanyl-D-alanine peptidase (at least at a 10 mM con- centration) but they behave as noncompetitive inhibitors of the Zn2+ D-alanyl-D-alanine peptidase. [ABSTRACT FROM AUTHOR]

The carbohydrate structure and complete amino acid sequence of a human lambda-type immunoglobulin light chain, protein Sm Z, has been determined. The protein was isolated from the urine of a patient with a plasma cell dyscrasia resembling γ-heavy-chain disease. 13 tryptic peptides covering the entire polypeptide chain of 135 residues were isolated from the aminoethylated protein, and 15 chymotryptic peptides, accounting for 131 residues, were recovered from the carboxymethylated protein. The sequence of 18 of these peptides was partially or completely determined by the Edman-dansyl technique or C-terminal analysis, permitting the establishment of the complete primary structure of the polypeptide chain. The sequence established that this light chain possessed an intramolecular deletion of 81 amino acid residues. The N-terminal 30 residues showed considerable homology with other lambda chains of subgroup II. The defect began at position 31, in the first hyper-variable region, and encompassed the remainder of the variable region through position 109. The constant region was fully intact and normal synthesis recommenced with a glutaminyl residue at position 110, the first residue of the constant region. This light chain contained carbohydrate in the hyperyanable region just preceding the deletion. The precise number and locations of the oligosaccharide chains were established by amino acid sequence analysis of glycopeptides isolated from proteolytic hydrolysates by chromatography on Bio-GeI P-6 columns. These studies showed that protein Sm A contains one N-glycosidically-linked chain attached to asparagine-25 and one O-glycosidically-linked oligosaccharide chain attached to serine-21. The structures of the oligo-saccharide chains were determined by methylation analysis, gas chromatography and hydrolysis with specific glycosidases. The structure of the N-glycosidically-linked chain was NeuAc(α2-→6)Gal(β1→4)GlcNAc(β1→2)- Man(α1→6)[NeuAc(x2→6)Gal(β→4)GlcNAc(β1→2)Man(α1→3)]Man(β1→4)GlcNAc(β1→4)[Fucα1→6]-GlcNAc→Asn. The second O-glycosidicalIy-linked chain was a disialylated tetrasaccharide with the structure, Neu(x2→3)Gal(β1→3)[NeuAc(α2→6)GalNAC→Ser. This mucin-type disialylated tetrasaccharide in close proximity to N-asparagine-linked chains has not been previously observed in the oligosaccharide chains of immunoglobulins. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of the u chain of the mitogenic lectin from Vicia sativa has been determined. The polypeptide was digested by trypsin and chymotrypsin. The fragments were isolated and most of them were sequenced by automatic solid-phase Edman degradation. A total sequence of 52 amino acid residues was obtained which is homologous to the α subunits of the lectins from Pisum sativum, Lens culinaris and Vicia faba. The central part (residues 13–32) in particular is completely conserved in all four proteins. In contrast to the three other known sequences, the V. sativa α subunit has an additional serine at the N terminus. The differences of the related amino acid sequences of these lectins and concanavalin A, the lectin from Canavalia ensiformis, have been compared using the relative substitution frequency found in homologous proteins by McLachlan. The degree of homology decreases in the order: V. sativa > P. sativum > V. faba > L. culinaris > C. ensiformis. Prediction of the secondary structure according to Chou and Fasman reveals that the lectin α chain is similar to concanavalin A in the first half of tile molecule whereas the C-terminal part apparently tends to form an α helix. [ABSTRACT FROM AUTHOR]

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a βαβ unit resembling that found in the dehydrogenases, Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter. [ABSTRACT FROM AUTHOR]

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28 000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complete sequence of the N-terminal half up to position 122. [ABSTRACT FROM AUTHOR]

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5-Pro have been used as models for the chromatographic investigation of lysine-peptide-oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix. Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the e-amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ϵ-amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides. Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation-fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of a brain-specific protein, S-100, has been determined by the analysis of four cyanogen bromide peptides and fourteen tryptic peptides derived from the protein. The protein chain consists of 91 amino acid residues and has a molecular weight of 10 507. The sequence shows regions of strong clustering of hydrophobic, basic and acidic amino acids, with an apparent calcium-binding site in the acidic cluster. [ABSTRACT FROM AUTHOR]

The quaternary structure of Escherichia coli K12 aspartokinase II — homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr = 176000. It is composed of two subunits having the same molecular weight and the same charge. The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine-containing peptides and the uniqueness of the N-terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II — homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I — homoserine dehydrogenase I. [ABSTRACT FROM AUTHOR]

Five intracellular proteolytic enzymes from Neurospora crassa were isolated and partially characterized: an acidic and an alkaline endopeptidase, one carboxypeptidase and two aminopeptidases. All these proteinases were purified from the same crude extract to homogenity by heat treatment, precipitation with ammonium sulfate, chromatography on DEAE-cellulose, CM-cellulose, DEAE-Sephadex, hydroxyapatite and by gel filtration. The acid proteinase hydrolysed acid-denatured haemoglobin at pH 3.0. The alkaline proteinase and the carboxypeptidase are serine proteinases that require a sulfhydryl group for activity. The aminopeptidases are both metallo-proteinases; one posseses broad specifity to the B-chain of oxidized insulin, the other posseses only narrow specifity and can only split the N-terminal basic amino acids of peptides. [ABSTRACT FROM AUTHOR]