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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-alpha, all-beta, alpha/beta and alpha + beta, 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.

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]

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]

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]

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]

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]

1. Adenylate kinase (ATP: AMP phosphotransferase) has been purified 490-fold from porcine muscle with a final yield of 60 mg/kg muscle. 2. The amino-acid composition is Asp11 Asn2, Thr14, Ser11, Glu19,, Gln6, Pro6, Gly19>, Ala6, Cyst2, Val17, Met6, Ile9, Leu18, Tyr7, Phe5, Lys21, His2, Arrg11. 3. The protein molecules a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine residue at the C-terminus. 4. Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α-protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained. 5. The primary structure of porcine adenylate kinase was given. [ABSTRACT FROM AUTHOR]

1. Tryptic digest, s of the [14C]carboxymethylated derivatives of the t, hree isoenzymes EE, ES and SS of horse liver alcohol dehydrogenase have been compared in "fingerprint" experiments. 2. Eight peptide spots present in the digest of the carboxymethylated EE enzyme were not detected in the digest of the carboxymethylated SS enzyme; and seven spots found in the latter were not discovered in the former. No other differences were noticed. The ES derivative yielded both types of spots but in reduced amounts, It is concluded that the E- and S-types of subunits are very similar and that the ES isoenzyme is a hybrid molecule. 3. From the carboxymethylated SS and ES isoenzymes the S-chain peptides that, differ from their counterparts in the E-chain were prepared and their structures analysed. They were compared to the known structures [ 1 — 3] of the corresponding E-chain peptides. It is concluded that all the differences between the two sets of peptides are accounted for by amino acid changes at only six positions along the protein chains of the E- and S-types of subunits, and an ancestral geneduplication is suggested. The differences at five positions (17, 94, 101, 110 and 366) are amino acid exchanges compatible with one-base mutations, while the nature of the sixth difference (position 115) is not fully established. 4. The six differences make the S-chain more hydrophobic and three units of charge more positive than the E-chain. These properties fit the solubilities and electrophoretic mobilities of the three isoenzymes. The difference in substrate specificity between the E- and S-chains might be explained by a direct participation in the substrate binding site of some of the residues exchanged, but, other explanations cannot be excluded. [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]

The light chain fraction was separated from chicken gizzard muscle myosin. After S-carboxymethylation or performic acid oxidation, two light chain components (20 000-M[subr] and 17 000-M[subr] chains) were isolated by chromatography on a column of DEAE-cellulose in the presence of 4 M urea. Tryptic peptides of the S-carboxymethylated 20 000-M[subr] chain were isolated, and their sequences were determined. The alignment of these tryptic peptides in the chain was deduced from the amino acid compositions and from the partial sequences of peptic peptides of the oxidized protein. The established sequence consists of 171 amino acids and its calculated molecular weight is 19692. Comparing the sequence with those of L-2 chains from chicken and rabbit skeletal muscle myosins, 81 and 78 amino acid substitutions were recognized, respectively, including insertions and/or deletions. [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 complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,χ) has been determined, thus completing the sequence determination of the entire heavy chain. The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions. [ABSTRACT FROM AUTHOR]

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physicochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 μg). [ABSTRACT FROM AUTHOR]

By conversion of prochymosin into active chymosin an N-terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology. [ABSTRACT FROM AUTHOR]

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

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

Our previous studies [1,2] on maleyl bovine αS1-B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH-terminal Tm peptide containing 48 residues was also reported. In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1-B casein. The αs1-B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex-50X2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1-B casein deduced from the analysis of Tm peptides. The three Tm peptides from αs1-B casek@ which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1-B ease@. The remaining three CN peptides from αs1-B casein for which identical fragments could not be located fun the Tm peptides, were digested with trypsin (in two eases after maleylation) and the resulting fragments were purified by Sephadex column chromatography. These trifle peptides provided the remaining gaps. This permitted in to locate the relative position of all the CN peptides in the αs1-B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1-B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1-B casein provided the missing overlaps. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

The complete sequence (153 amino acids) of horse heart myoglobin has been established. The sequence of a peptide isolated from chymotrypsin hydrolyzates of globin allowed to fill a gap in the tentative sequence previously proposed; this peptide was studied by means of pepsin hydrolysis, action of N-bromosuccinimide on histidyl bonds, and Edman degradation method associated with dansylation. After hydrolysis of globin by chymotrypsin treated by TLCK, the same major peptides were found as after hydrolysis by non-treated chymotrypsin. These peptides were identified by ionexchange resins and bidimensional paper chromatography, and also by the determination of their amino-acid composition. The suppression of secondary cuts explains the presence of new peptides, whose the composition and N-terminal Sequences were determined, it was then possible to confirm several sequences which could be previously deduced from the study of split products after cyanogen bromide treatment. The specificity of chymotrypsin towards certain types of peptide bonds is discussed. Horse myoglobin, as compared with sperm whale myoglobin, contains 18 differences: 17 substitutions and one inversion. The major part of the substitutions are located ih N- and C-terminal sequences: they are all punctiform, resulting from the replacement of only one base in each codon. [ABSTRACT FROM AUTHOR]

Using automated stepwise Edman degradation of suitable overlapping peptides, the complete amino acid sequence of the 268 residue peptida α1-CB7 from calf skin collagen was determined. The preparation and ordering of the chymotryptic, tryptic and thermolytic peptides is described in the preceding paper. As shown previously for other peptides from the helical region of collagen, glycine appears in every third position and proline is present in high amount. Hydroxylation of proline to 4-hydroxyproline and lysine to hydroxylysine only occurred in the Y position of the tripeptide unit Gly-X-Y. Non-random distribution of other amino acids between the X and Y positions was also observed. [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 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]

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]

Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactively labelled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actins present in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic actin present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 105 cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity. [ABSTRACT FROM AUTHOR]

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

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 arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-l-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent from digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation of the peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains. [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 two halves of the ATPase, Mr, 115000, from sarcoplasmic reticulum produced by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr 60000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55000 by gel filtration. The two halves of the 60000 Mr fragment (Mr 33000 and 24000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphatc. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated. [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]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B 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 purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, 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 tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]

The two rat proinsulins have been separated and the amino acid compositions determined. Using a method previously described for preparing C-peptides from ox and pork pancreases, three peptides were isolated from rat pancreases and the amino acid compositions of these peptides determined. Data are presented which strongly indicate that two of these peptides constitute the C-peptide part of the connecting peptide of the two rat proinsulins. [ABSTRACT FROM AUTHOR]

Purification of acid phosphomonoesterase from Phaseolus mungo seedlings is reported here. Phosphatase homogeneity was evaluated at 85-90% by several methods. The enzyme is a single polypeptide chain of molecular weight 55000 ± 5000 daltons with serine and leucine as the N and C terminal residues, respectively. A saccharide fraction containing 8% of protein weight as reducing sugar and 3% as amino sugars was found. Attempts to dissociate the saccharidic and protein moieties were unsuccessful. It was concluded that a single phosphatase was active toward phosphomonoesters, phosphoanhydride, and β-D-glycosyl 1-phosphate. [ABSTRACT FROM AUTHOR]

Two N-terminal fragments of α(A)-chain and β(B)-chain in human fibrinogen have been isolated from a plasmic hydrolyzate. The fragment from the α(A)-chain consisted of 43 amino acid residues including two half-cystine residues. On treatment with thrombin, this fragment produced two other peptides in addition to fibrinopeptide A and its analogues. One was a tri-peptide, Gly-Pro-Arg, and the other a peptide containing 24 amino acid residues having N-terminal valine. The partial amino acid sequence of the α(A)-chain has been found to be: Ala-Asp- Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-GIu-Arg-I{is- Gln-Ser-Ala-Cys-Lys.Asp-Ser-Asp-Trp-Pro-Phe-(Cys-Ser-Asp-Glu-Trp-Asn-Tyr)-Lys. The fragment from the β(B)-chain consisted of 21 amino acid residues. This fragment released fibninopeptide B on treatment with thrombin. The amino acid sequence of the β(B)-chain fragment is: Pyr-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Asp-Lys. The linkages between fibrinopeptides and fibrin, which are rapidly hydrolyzed by thrombin, are very resistant towards plasmin. [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]

1. The perturbation of the spectrum of free tyrosine that occurs when this amino acid is combined to other amino acids or in the form of acetyl derivative, acetyl derivative amide or acetyl derivative ethyl ester was studied. 2. The difference spectrum of each compound was obtained by measuring the absorbance of its solution against that of a mixture of its component free amino acids. 3. A red-shift of the 2,400 Å absorption band, in 0.1 M NaOH, or 2,230 Å, at pH 7, was clearly detected. 4. Maximum perturbation is produced when tyrosine has both its amino and carboxyl groups involved in peptide bonds. [ABSTRACT FROM AUTHOR]

Proteose-peptone component 8F (or ‘8-fast’) has been prepared from bovine milk. Sedimentation equilibrium analysis, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and gel filtration in urea-containing buffers all gave molecular weight values between 3300 and 3900. The N-terminal sequence was found to be Arg-Glu- by dansylation and Edman degradation. Hydrazinolysis released lysine from the C-terminus. A mixture of carboxypeptidases A and B showed that the C-terminal sequence was -Thr-(Arg,Ile,Asn)-Lys. The phosphate content was 3.8 mol/mol and was completely released by a short alkaline hydrolysis indicating linkage to serine. This and all other aspects of the composition were entirely consistent with the identification of this proteose- peptone as residues 1–28 of the β-casein molecule. This identity was confirmed by a peptide mapping procedure. Thus proteose-peptone component 8F represents the N-terminal fragment when the γ1-caseins are formed by proteolysis of β-casein. [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]