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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]

The purification and the amino acid sequence of the peptide containing the essential sulphydryl group of arginine kinase from Homarus vulgaris muscle is described. The fractionation of the tryptic digest of arginine kinase labelled with N-[1-14C]ethylmaleimide has been caused out using gel filtration, ion-exchange chromatography and paper chromatography. The composition of the radioactive peptide, isolated from the digest, is reported together with its amino acid sequence. A structural comparison is made between the peptide of arginine kinase and the corresponding active-cysteine containing peptide isolated from rabbit muscle creatine kinase. [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [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 digestion of peptide A, the C-terminal fragment of thioredoxin, obtained by cyanogen bromide cleavage, resulted in the isolation of nine peptides. The complete amino acid sequence of each was determined, and the N-terminal and C-terminal fragments of peptide A were identified. Together the nine tryptic peptides accounted for all 71 residues of peptide A. Hydrazinolysis and studies with carboxypeptidase A established -Asn-Leu-Ala to be the C-terminal sequence of peptide A and of thioredoxin. [ABSTRACT FROM AUTHOR]

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

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [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]

With the aim of establishing a starting point for the determination of the complete amino acid sequence of the major parvalbumin from hake muscle, the peptides resulting bom the tryptic digestion of the S-sulfo-derivative and of the performic, acid-oxidized protein were examined. A total of 20 peptides were thus obtained m sufficient quantity and m a state of purity adequate for further studies. Their complete or partial sequence is reported. When due account is taken of obvious overlaps, the total of these peptides fully accounts for the composition of the whole molecule with the exception of one alanine residue, and for the number of peptides expected from the presence of 12 lysines and 1 arginine in this parvalbumin. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [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]

This article is the sixth and last of a series devoted to the primary structure of bovine β-casein [1–5]. In the preceding paper, we presented a partial sequence of this protein in which 156 out of the 209 amino-acid residues were exactly positioned [5]. Only a few data were given on the tryptic peptides T2 and T3 [1] which corresponded to the unknown part of the molecule. In the present communication, we establish the amino-acid sequence of these 2 peptides, and discuss the salient features of the β-casein molecule. Bovine β-casein A2 is a phosphoprotein constituted of a single polypeptide chain containing 209 amino-acid residues. Among them are five phosphoseryl residues. From sequence data, it can be written: Asp4, Asn5, Thr9, Ser11, SerP5, Glu17, Gln22, Pro35, Gly5, Ala5, Val19, Met6, Ile10, Leu22, Tyr4, Phe9, Trp, Lys11, His5, Arg4. Its exact molecular weight is 23982, a little higher than that of αs1-casein, which is 23616 [6]. The total number of 27 amide groups (Asn + Gln) agrees well with previous data obtained by Pion et al. [7] and Peterson et al. [8]. The average hydrophobicity, calculated according to Bigelow [9], is 1330 and it places β-casein among the most hydrophobic proteins. The sequence obtained emphasizes the uneven charge distribution along the peptide chain. A high negative net charge is located in the first 50 residues. The high proline residue content (16.7%) and the rather uniform repartition of this residue exclude the possibility of a high α-helix content. Furthermore, the complete absence of segments containing more than 2 adjacent prolyl residues seems to preclude the possibility of poly-L-proline structure that was expected from optical-rotatory-dispersion studies [10]. An interesting extension of the present work is the discovery by Gordon et al [11] that γ-, TS-, R- and S-caseins are fragments of β-casein likely originating from tryptic-like cleavages of the peptide chain next to the carbonyl groups of some lysyl residues. The γ-, TS. A2- and S-, TS. B- and R-caseins respectively represent residues 29 to 209, 106 to 209 and 108 to 209. These results explain the occurrence of only one phosphorus atom in the γ-caseins, the absence of phosphorus in the TS-, R- and S-caseins [12]. Localization of the substitutions which differentiate the known variants of bovine β-casein also explains the relationship between β-, γ-, R-, S- and TS-caseins [12,13]. The NH2-terminal tryptic phosphopeptide, previously analyzed by Peterson et al. [14], has been sequenced independently by Manson and Annan [15]. Their results are identical to those reported in a preceding paper [4]. [ABSTRACT FROM AUTHOR]

Previous reports [1-5], we have described some of the features of the primary structure of bovine αs1-casein. In the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed. In the polyeptide chain, the region containing the phosphopeptides Tm1 (T1-T2), Tm1T2 and Tm1T1 [1,4,5], had only been slightly studied as yet. Because of the difficulties encountered in the breakdown of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides. It was confirmed that peptide Tm1T2 contains three hydroxy-amino acids (2 Ser, 1 Thr), but, instead of three [1], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues m this peptide are O-phosphorylated. The remaining gap in the sequence of the central part of peptide Tm1T2 was bridged by studying two fragments obtained by papain digestion of the dephosphorylated preparation. Instead of four serines, postulated earlier from the results of the amino-acid composition of peptide Tm1T1 [1], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be ready removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be O-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide Tra1T1 were further degraded using classical methods for the determination of the amino acid sequence. In the light of all the results obtained on this phosphoprotein (αs1-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [1-5], and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp7, Asn8, Thr5, Ser8, SerP8, Glu25, Gln14, Pro17, Gly9, Ala9, Val11, Met5, Ile11, Leu17, Tyr10, Phe8, Lys14, His5, Trp2, Arg6, indicates that there is a higher number of acidic than basic residues in this protein. On the basis of the intrinsic dissociation constants of titratable groups in proteins [6], the negative net charge of the molecule was estimated to be 22 at pH 6.5 and 28 at pH 8.6. Since Bigelow's parameter for thc average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein The high amount (8.5%) and uniform distribution of prolyl residues indicate that this protein has limited structuralcoiling possibilities. The polypeptide chain contains three hydrophobic regions, viz. 1-44, 90-113 and 132-199. The first two are characterized by the fact that basic residues predominate over acidic residues. The third region, where most of the aromatic residues are located, contains very few basic residues and is therefore of more acidic character. Two regions, 45-89 and 114-131, are hydrophilic. The former contains more than half of the total acidic residues, in particular seven of the total eight phosphoseryl residues. Another remarkable feature is the concentration of amino acids with carboxylic side chains, particularly glutamic acid, in the vicinity of two clusters of phosphoseryl residues. It may be noticed that the phosphoseryl residues m αs1-casein are arranged m a manner similar to those in β-casein [8]. In particular, in αs1-casein, the 62-70 region containing four phosphoseryl residues, is similar to the 13-21 region in β-casein [8]. It is interesting to point out that ail but one of the phosphoseryl residues always occur m position n with relation to a glutamyl or a phosphoseryl residue, which is in position n + 2. From this, it would appear that there is an enzymatic phosphorylation of the polypeptide chum (carried out by a phosphoryl kinase which may require a negative charge in the phosphorylation site) rather than a direct incorporation of phosphoserine into the polypeptide chain during protein synthesis. The location of the amino-acid substitutions that characterize the αs1-Casein D and the β-casein C variants has provided evidence for an enzymatic phosphorylation of casein [9]. The problem of the phosphorylated sites in αs1- and β-caseins will be discussed in a forthcoming paper. The repeating amino-acid sequences located in the two regions of αs1-casein, viz. 70-84 and 110-125, are also of interest. The primary structure of αs1-casein given here is that of the genetic variant B. The three other variants A, C and D have also been studied. The C and D variants differ from the B variant by the substitutions of Gly/Glu in position 192 [10] and ThrP/Ala in position 53 [9], respectively. The A variant is characterized by a deletion of thirteen amino-acid residues from position 14 to position 26 in the hydrophobic NH2-terminal part of the polypeptide chain [11]. Because of this deletion, there m an important change in the physico-chemical properties of αs1-casein [12, 13]. Some important features of the amino-acid-sequence determination should be noted. Firstly, the great value of maleic anhydride as a reversible-blocking reagent for amino groups of proteins [14], and the usefulness of thermolysin as a specific enzyme for degrading polypeptides [15]. Secondly, the difficulties encountered during the study of the phosphopeptides: these peptides were difficult to purify since they have similar anionic characters and only react slightly with ninhydrin during paper revelation; other difficulties have been the high destruction of phosphoserine during hydrolysis with HCl 5.7 N, the poor yield in the substractive Edman degradation when the NH2-terminal residue is a phosphoserine, and the failure of endopeptidases and exopeptidases to split off peptide bonds that are close to phosphate groups. Fortunately, alkaline phosphatase has proved to be a useful tool for removing phosphate groups, thus avoiding most of these difficulties. Since the amino-acid sequence of αs1-casein is now known, the degradation of this protein by coagulating and other proteolytic enzymes during cheese ripening may be studied. In particular, bitter peptides originating from αs1-casein may be located. As we have already briefly noted [4], it is obvious that one of the three bitter peptides isolated by Matoba et al. [16] from a tryptic hydrolysate of whole casein, was the segment 23-34 of the polypeptide chain of αs1-casein. In addition, the knowledge of the primary structure will be of help in understanding many aspects of the relations between structure and physical properties in this phosphoprotein. [ABSTRACT FROM AUTHOR]

This paper is the fifth of a series devoted to the primary structure of the bovine β-casein A². The first two communications dealt with the isolation, analysis and positioning in the peptide chain of the tryptic and “CNBr” peptides [1,2]. The last two described the sequence of 65 residues from the carboxyl-terminus and of 32 residues from the amino-terminus [3,4]. We report here a partial sequence which includes 156 residues (out of the 209 residues of the β-casein A²) exactly positioned in the peptide chain. Two segments including 53 residues (from position 36 to 68 and from 73 to 92) remain to be analyze. In addition to the tryptic and “CNBr” peptides originating from β-casein A², two peptides issued from a tryptic digest of β-casein B were used as starting material. The position of the substitution His→Gln which differentiates the β-casein A² from the β-casein A³, already given in a preliminary communication [5], is indicated, as it is for the substitution Ser→Arg which represents one of the two differences occurring between the β-casein A² and the β-casein B. Four of the five phosphorus atoms of the β-casein A² have been previously located in the NH2-terminal tryptic peptide [4]. The fifth has been located at position 35. All the phosphorus atoms of the β-casein A² occur as phosphoserine. The complete structure of the β-casein A² will be given and discussed in the next and last paper. [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 primary structure of the glycopeptide from the posterior lobe of the pig pituitary is: Ala- Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu. The carbohydrate is linked to the peptide chain via the asparagine residue. [ABSTRACT FROM AUTHOR]

The globin of sheep heart myoglobin was first cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration and preparative paper electrochromatography; each fragment was submitted to endopeptidase digestion and the complete amino acid sequence has been determined. The intact globin and the median cyanogen bromide peptide were analysed by the protein sequanator (Edman). This analysis confirmed the sequence of the first 46 N- terminal residues of the intact globin and established the sequence of the first 39 N-terminal residues of the median cyanogen bromide peptide. Between sheep and beef myoglobin six differences over 153 residues can be noticed; among the six amino acid replacements, two correspond to the exchange of two bases and four to the exchange of one base in the coding triplets. [ABSTRACT FROM AUTHOR]

The primary structure of sheep para-ℵA-casein, the N-terminal portion of ℵ-casein, was established. The reduced alkylated protein was subjected to digestion with trypsin. The resulting soluble peptides were purified by a combination of Dowex I X 2 chromatography and electrophoresis and chromatography on paper; the core peptides were solubilized in 50 % formic acid and filtered on Sephadex G-50. The amino-acid sequence of these peptides was determined chiefly with a Sequencer. Alignment of thc tryptic peptides into a single chain containing 105 amino acids was determined, from basic overlap peptides (tryptic core peptides with several basic amino acids; chymotryptic peptides). Some comparisons were achieved with cow para-ℵA-casein. [ABSTRACT FROM AUTHOR]

The isolated α-chain of sea anemone collagen was cleaved at methionyl residues with cyanogen bromide. Eleven unique peptides were isolated by ion-exchange and molecular-sieve chromatography, eight of them in approximately equimolar amounts. Characterization of the eleven peptides with regard to amino-acid composition and molecular weight demonstrated that the isolated peptides account for all of the amino acids and for the molecular weight of the α-chain within experimental error. Since the molecule of sea anemone collagen contains ten methionyl residues, the data of the present paper confirm earlier analytical and preparative results that this invertebrate collagen is made up of three identical α-chains. In comparison with known collagens, on the basis of quantitative and qualitative features of amino-acid compositions, the cyanogen bromide peptides of sea anemone collagen indicate genetical relationship of sea anemone collagen to basement membrane collagen. [ABSTRACT FROM AUTHOR]

The peptide side chains of the peptidoglycan of the Pseudomonas aeruginosa cell envelope have the sequence H2-L-AIa-GIu-A2pm-D-AIa-COOH. The D-glutamate is linked to the meso2,5-diaminopimelate (A2pm) by a peptide bond formed by the C-5 carboxyl group of D-glutamate. The C-1 carboxyl group of the glutamate does not occur in the amidated state in the cell wall. The configurations of alanine, glutamate, and 2,5-diaminopimelate have been confirmed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His -Ser -Asp -Ala -Val -Phe -Thr -Asp -Asn -Tyr -Thr -Arg -Leu -Arg -Lys -Gln -Met -Ala -Val -Lys -Lys -Tyr -Leu -Asn -Ser -Ile -Leu -Asn -NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N-terminus, are Identical to those in the corresponding positions in both porcine glucagons and secretin. The residues in positions 3, 12, 13, 14, 23 are identical to those in secretin, but not in glucagons, and position 10 is occupied by a tyrosyl and position 28 by an asparanginyl residue, like in glucagons but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagons, are taken into consideration then the similarity between these three polypeptide is still more evident. At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin-pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known. The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its X-terminal cyanogen bromide heptadecapept.ide that are susceptible to cleavage with trypsin. [ABSTRACT FROM AUTHOR]

The neurohypophysial hormones of a chondrostean, the sturgeon (Acipenser sp.), have been purified by adsorption onto neurophysin, dissociation of the complex hormone-protein by trichloroacetic acid precipitation and isolation of active peptides, from the supernatant solution, by paper chromatoelectrophoresis. Arginine vasotocin has been characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties as well. The amount of arginine vasotocin (about 50 nmol per g pituitary powder) is intermediary between those found for bony fishes (about 1000 nmol/g) and cartilaginous fishes (about 5 nmol/g). A second hormone, which can be classified in the oxytocin-like type by its electrophoretic migration and its pharmacological properties, has been disclosed. The very weak amount did not allow chemical identification. However the chromatographic behaviour and the pharmacological "profile" indicate that this hormone differs from the six known oxytocin-like peptides. [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 αA2 chain of bovine α-crystallin was fragmented by means of cyanogen bromide treatment and by tryptic, chymotryptic and thermolytic digestions. Twenty tryptic peptides were obtained from the S-aminoethylated αA2 chain, accounting together for the complete sequence. The direct Edman degradation and the dansyl-Edman technique were used to determine the sequences of the tryptic peptides. The order of the tryptic peptides was deduced from overlapping peptides obtained by cyanogen bromide treatment, tryptic digestion of the maleylated chain and chymotryptic and thermolytic digestion of the S-aminoethylated chain. The sequence of the αA2 chain comprises 173 residues and corresponds to a molecular weight of 19832. [ABSTRACT FROM AUTHOR]

Commercial lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963. Reduced and alkylated component IV was hydrolyzed with trypsin and the resulting peptides were separated by ion exchange chromatography on Dowex 50 X2 or gel filtration on Bio-Gel P-6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both. Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequence of six of these peptides was determined, using classical methods. When allowance is made for the occurrence of two homologous peptides, presumably resulting from heterogeneity m the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein. In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy-terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported. [ABSTRACT FROM AUTHOR]

A method has been elaborated for the large scale isolation of outer membranes of beef liver mitochondria. From these monoamine oxidase was partially purified and digested with trypsin and chymotrypsin to yield peptides containing riboflavin covalently linked to the peptide chain. A pure pentapeptide of riboflavin-5'-phosphate has been obtained by a series of chromatographic procedures. Edman degradation, followed by dansylation, revealed the amino acid sequence: Ser-Gly-Gly-X-Tyr, X being the amino add to which the flavin is attached via the 8α-carbon of the riboflavin. This site of attachment is concluded on the basis of analysis of the optical spectra in the neutral and cationic forms of the flavoquinone and of the electron spin resonance spectrum of the free radical cation. [ABSTRACT FROM AUTHOR]

Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase is almost completely inhibited by reaction of 4 moles of pyridoxal 5'-phosphate per mole of subunit chain of holoenzyme. Reduction of the pyridoxal 5'-phosphate inactivated holoenzyme with NaBH4 followed by carboxymethylation of thiol groups, and subsequent tryptic digestion, allowed the separation of two strongly fluorescent peptides. These have been identified as peptides T19 + T20 (residues 184 to 192) and peptides T22 + T23 (residues 198 to 216) of the sequence published by Harris and Perham for porcine enzyme, in which lysines 191 and 212 are blocked by pyridoxal 5'-phosphate Evidence for other lysyl residues reacting to a lesser extent with the reagent is reported among such residues, only lysine-2 (contained in the N-terminal peptide) has been unambigously identified. No reaction takes place at lysine-183. The homology between the sequence around lysine-212 and the sequence around the essential lysine residue in glutamate dehydrogenase is stressed. The role of pyridoxal 5'.-phosphate reactive lysines in the mechanism of glyceraldehyde-3--phosphate dehydrogenase is discussed. [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]

A series of peptides possessing the structure (L-Tyr-L-Ala-L-Glu)n, with n = 1, 2, 3, 4, 7, 9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N-hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7, 9, 13 were prepared by a polycondensation technique, followed by gel filtration. The circular dichroism spectra of the above oligopeptides were measured in the wavelength range 200-330 nm in a solution of 0.15 M sodium chloride -0.02 M sodium phosphate, pH 7.4, conditions at which the high molecular weight polytripeptide (Tyr-Ala-Glu)n exists in a helical conformation. The circular dichroic spectrum of the high molecular weight polymer exhibits two negative ellipticity bands: an intense band at 220 nm ([θ]max = 8700) and a weak band at 273 mn ([θ]max = 360). The tripeptide (n = 1) exhibits a positive ellipticity band at 227 nm ([θ]max = 4000) and a broad, positive, weak hand at 270 nm ([θ]max = 115). The other oligopeptides (n = 2 to 13) exhibit positive bands in the 227 n.m region, with ellipticity values that decrease as the degree of polymerization increases. Except for (Tyr-Ala-GIu)13, which has a negative peak centered at 216 nm, circular dichroic spectra of the other oligomers (n = 1 to 9) in the 200-210 nm region resemble that of random coils. In the 260-290 nm region, the circular dichroic curves of the oligopeptides gradually approach the 272 nm band of the polypeptide (Tyr-Ala-Glu)n, as the degree of polymerization increases. Under physiological conditions, only the (Tyr-Ala-Glu)13 shows an indication of helical content. [ABSTRACT FROM AUTHOR]

Presents an investigation of the sequencing of 65 amino acid residues at the carboxyl-terminus. Use of methods such as enzymic or chemical cleavages of the peptide chain; Fractionation of peptides by Sephadex or Dowex column chromatography, paper elctrophoresis or chromatography.

The substrate specificity of the sorghum acid protease was elucidated using synthetic peptides and pancreatic ribonuclease A as substrates. These studies revealed that the enzyme has a rather narrow and novel specificity requirement. The protease specifically cleaves the peptide linkages involving the α-carboxyl group of either aspartic acid or glutamic acid with the release of the acyl portion of these acidic amino acids. Further, the side chain carboxyl groups of aspartic acid arid glutamic acid in the peptides should be unsubstituted for the enzyme to act on the peptide, and therefore, glutaminyl and asparaginyl peptides are not cleaved by the enzyme. Studies carried out with performate oxidized ribonuclease and ribonuclease methyl esters have further substantiated the results obtained with the synthetic substrates. These results suggest that the sorghum acid protease will be very valuable in sequence analyses of proteins. [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]

1. Tryptic hydrolysates of denaturated glutamic-aspartic transaminase fractionated on a Sephadex G-25 column, gave an elution diagram containing six main fractions. When peptide maps were developed from the transaminase digest 37 spots were detected on the paper sheet. 2. It was demonstrated that the transaminase fractions (groups of peptides) as well as some of the individual peptides eluted from the map spots, possessed immunological activity. This was proved by their capacity to interact with rabbit pig heart antienzyme and rabbit antiserum against the pig-heart whole tryptic transaminase hydrolysate as well as to inhibit the homologous transaminase-antitransaminase reaction. Higher immunological reactivity was found among peptides moving faster in electrophoresis and chromatography. 3. The catalytic activity of rabbit transaminase (heart, liver, skeletal muscles), was inhibited to a substantial degree by the rabbit anti-pig heart transaminase. The above enzymes however, as was otherwise expected, gave negative precipitin reactions with antitransaminase. A comparative study of the reactivity between transaminases from pig, ox and rabbit and rabbit anti-transaminase against pig and ox heart enzymes as well as against pig-heart whole tryptic transaminase hydrolysate is presented here. [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]

Peptide A, the C-terminal cyanogen bromide fragment of thioredoxin, was degraded with chymotrypsin and pepsin. Partial sequences of 12 chymotryptic and 6 peptic peptides and the N-terminal tryptic peptide of trifluoro-acetylated peptide A were determined. The results were used to establish the order of the previously described tryptic peptides of peptide A and lead to the complete amino acid sequence of peptide A. Previous experiments had established the amino acid sequence of peptide B, the N-terminal cyanogen bromide fragment of thioredoxin and the present results thus give the complete amino acid sequence of thioredoxin from Escherichia coli B. The molecule contains 108 residues in a single polypeptide chain with a molecular weight of 11,657 as calculated from the sequence. The functional group of the protein occurs in position 32 to 35 and consists of a disulfide bridge formed by two half-cystine residues separated by a glycine and a proline residue. No metals were found as part of the functional group. [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]

Chymotryptic cleavage of α1-CB6, the C-terminal CNBr peptide of the α1-chain from calf skin collagen, yields three peptides, designated C1 (positions 1-113), C2 (positions 114-196) and C3 (positions 197-217), respectively. The present paper describes the amino acid sequence of C2. It was determined exclusively by application of the automated Edman degradation. The entire peptide C2 as well as some of its tryptic and thermolytic peptides were subjected to this procedure. The helical portion of α1-CB6-C2 (positions 114-192) was found to consist of tripeptide units containing glycine in every third position, hence being typical of the collagen structure. The α1-chain of calf skin collagen contains a 3-hydroxyproline residue which was located in position 164 in the tripeptide unit Gly-3Hyp-4Hyp. This is the first case of a vertebrate collagen in which hydroxylation was found of a proline in position X of the tripeptide unit Gly-X-Y. The helical part of the α1-chain terminates in a five-fold repetition of the tripeptide Gly-Pro-Pro. The four C-terminal amino acids of C2 are already part of the nonhelical area at the end of the α1-chain which in addition comprises the entire peptide C3. [ABSTRACT FROM AUTHOR]

In order to ascertain the physiological role of the peptides which enhanced, the growth of Streptococcus thermophilus in milk, fourteen stimulating peptides were isolated and purified and their qualitative amino-acid composition was determined. The purification process was the following: after hydrolysis at pH 7.5 of whole casein by the neutral protease from Micrococcus caseolyticus the stimulating peptides were fractionated by gel chromatography (Bio-Gel P6), by column chromatography on cation-exchange resins AG 50W X2 (0.05 M pyridine-formate buffer at pit 3.10 to 1.5 M pyridine-acetate buffer at pH 5:10). Further purification was made by column chromatography on anion-exchange resins AG1 X2 (0.1 M pyridine-acetate buffer of pH 7.5 to 6 M acetic acid with a concave gradient system) and by paper chromatography (solvent: butanol-acetic acid-water, 4:1:5, v/v/v) and high tension electrophoresis (30 min; 60 V/cm). Gel filtration of enzymic digest of the whole casein led to six peptide fractions. Three fractions were stimulatory and exhibited a mean molecular weight of (B) 2500, (C) 1500, (D) 1000. Fraction C was the most stimulatory. Three other fractions were inhibitory and contained the highest molecular-weight peptides (about 5000) and peptides with high contents of aromatic residues. Ten and twenty peptide fractions were isolated respectively from Fractions B and D by chromatography on ion-exchange resins; but the peptides obtained after further purification were no more stimulatory. Eleven stimulatory and two inhibitory fractions were separated from Fraction C by column chromatography on cation-exchange resins. Purified stimulatory peptides were obtained from two fractions (C1 and C42) only, after anion-exchange resins (three peptides from Fraction C42, eleven peptides from Fraction C1). These fourteen purified stimulatory peptides display a very variable qualitative amino-acid composition. The polar part (Lys, Glx, Asx) was as important as the hydrophobic part (Pro, Leu, Val, Ala, Gly). The number of distinct amino-acid residues from one peptide was also quite variable: fourteen to four residues. Only lysyl residue was present in all peptides and tryptophan was not present. Three peptides that contained cysteine were issued from %-casein [18]; two peptides (C1a2α and C1C3) could be located respectively to Ala26…Gln22… and Ile26… Arg119 regions from bovine αs1B casein. A complete mixture of amino acids exerted growth stimulation. The set of the amino acids present in a stimulatory peptide was always stimulatory. These data substantiate the following conclusions: (a) stimulatory peptides acted as amino acid source; the amino acids have to be supplied in specific proportions in order not to disturb the cellular amino acids pool and the regulation of amino acid synthesis by Str. thermophilus; (b) the requirements in the peptide-length residues and lysyl residue could be explained by a control system of peptide intake, for example, by a peptide-transport system that would differ from that described in Escherichia coli. [ABSTRACT FROM AUTHOR]

α-Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH-4.0 precipitation, followed by gel filtration with Sephadex G-100. A final purification stage using DEAE-cellulose was necessary in some preparations. Peptides derived from the reduced, S-aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypain, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high-voltage paper electrophoresis. From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α-laotaIbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α-Iactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [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]

In α-crystallin from the nuclear part of the lens, polypeptide chains were found, which were not present in the cortex of the lens. These additional subunits were fractionated by means of chromatography on DEAE-cellulose (DE-Q) or CM-cellulose (CM-Q). Characterization of two of them showed that they were identical to shorter αA-chains, and hence they were designated as αA21-151 and αA11-151. By studying α-crystallin as function of fiber cell development it turned out that the subunit structure of α-crystallin is not constant. The polypeptide chains in the α-crystallin aggregates are subject to specific and limited degradation processes, causing an ever changing polypeptide chain composition. [ABSTRACT FROM AUTHOR]

The amino-acid composition of a polypeptide fraction that contains a few closely related polypeptides was determined. These polypeptides are obtained in high yield from red kidney beans and stimulate RNA synthesis in a bacterial system and in mouse spleen lymphocytes. Peptide maps of the carboxymethylated fraction were prepared and tryptic peptides purified and analysed for total composition and amino acid sequence. The results revealed that the differences between the polypeptides are due to a few microheterogeneities in the form of amino acid exchanges, but no evidence for the existence of polypeptides of different size or overall structure were obtained. It is concluded that the purification method yields different types of highly similar polypeptides with a molecular weight in the range of 10000, corresponding to about 80 residues, and an unusually high cysteine content of 19 mol/100 mol. These facts show that the present polypeptides are different from all previously analysed factors stimulating RNA synthesis. [ABSTRACT FROM AUTHOR]

Some techniques, facilitating the determination of the amino-acid sequence of proteins or protein fragments available in 10 nanomole amounts are presented. Fluorescamine was used to follow the purification of proteins and peptides without ineterfering in the determination of the amino-acid composition and sequence. These techniques were applied on the coat protein of coliphage MS[SUB2] and on some peptides obtained from a chymotryptie digest of the the assembly protein of MS2. [ABSTRACT FROM AUTHOR]

Describes reaction of glutamate dehydrogenase with glyoxal and NaB[sup 3]H[sub 4] and the labelled protein digested with trypsin. Isolation and identification of radio peptides in the primary structure of the enzyme; Small amounts of radioactivity contained in many peptides; Unspecific reactions with glyoxal; Identification of lysine-27 as the residue reacting with glyoxal; Conclusion that lysine residues.