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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]

The article presents a list of articles related to biochemistry. The articles include "The Reaction of Hemin With H2O2," by M. L. Kremer, "Amino Acid Sequence of the Monomer Subunit of the Giant Multisubunit Hemoglobin From the Earthworm Pheretima sieboldi," by T. Suzuki, "Purification and Properties of Saccharomyces cerevisiae Acetolactate Synthase From Recombinant Escherichia coli," by C. Poulsen and P. Stougaard, "The Mitochondrial Inner-Membrane Anion Channel Possesses Two Mercurial-Reactive Regulatory Sites," by A. D. Beavis.

Presents several research papers related to biochemistry. "Effect of Thiol Reagents on the Proton Conductivity of the H+ATPase of Mitochondria," by F. Guerrieri and S. Papa; "Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae," by R. Hakenbeck and M. Kohiyama; "Reutilization of Preformed Pyrimidines for the Synthesis of DNA in Rat Liver and Kidney," by J. Seifert; "Human Progastriesin: Analysis of Intermediates during Activation in Gastriesin and Determination of the Amino Acid Sequence of the Propart," by B. Foltmann and A. L. Jensen; Others.

Presents a list of studies that will be published in the "European Journal of Biochemistry." "Peroxisomal Oxidation of L-2-Hydroxyphytanic Acid in Rat Kidney Cortex," by J.-P. Draye, F. van Hoof, E. de Hoffmann and J. Vamecq; "Probing the Heparin-Binding Domain of Human Antithrombin III With V8 Protease," by C.-S. Liu and J.-Y. Chang; "Protein Phosphatases of the Guinea-Pig Parotid Gland," by G. Mieskes and H.-D. Söling; "Amino Acid Sequence of Endothiapepsin--Complete Primary Structure of the Aspartic Protease From Endothia Parasitica," by V. Barkholt.

The amino acid sequence of the light chain of a human monoclonal IgA1 (Mem) was established, in part by analogy with already known sequences. By homology its variable part was shown to belong to the VλI subgroup while the isotype-associated amino acid residues characterized it as Mcg+, Kern+ and Oz-. The normal primary structure of this chain was in contrast to its abnormal physical and antigenic properties: (a) its apparent molecular mass estimated by SDS/polyacrylamide gel electrophoresis, by gel filtration chromatography and by gradient ultracentrifugation was found to be lower by &assymp; 10% than the values (23.5 kDa) of ‘normal’ light chain used as controls; (b) the λI chain Mem, when tested in native state was not antigenically reactive. These abnormalities were reverted when the chain was treated with 8 M urea. These data suggest that the abnormal behaviour of λI chain Mere is at a conformational level. [ABSTRACT FROM AUTHOR]

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [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]

Incubation of calf tens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different β-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-γ-glutamylyltransferase, EC 2.3.2.13). One of these is βBp, the predominant subunit of β-crystallin, of which the amino acid sequence is known. The site of amine-labeling in βBp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled β-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled β subunits. [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]

Porcine secretin is a heptacosapeptide with the amino acid sequence His-Ser-Asp-Gly-Thr- Phe -Thr- Ser -Glu -Leu -Ser -Arg -Leu -Arg -Asp-Ser -Ala -Arg -Leu -Gin -Arg -Leu -Leu-Gln-Gly-Leu- Va1NH2. Fourteen of its twenty-seven amino acids occur in the same position as they do in porcine glucagon. [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]

Two c-type cytochromes were isolated from cells of the gram-negative bacterium Aquaspirillum itersonii grown under low aeration in the presence of nitrate. The major component, cytochrome c-550, was equated with the (single) c-type cytochrome previously reported to be present in this organism [Clark-Walker, G. D. & Lascelles, J. (1970) Arch. Biochem. Biophys. 136, 153-159], although a significantly higher molecular mass was apparent in the present work. The complete amino acid sequence of this cytochrome is reported in the accompanying paper. A second soluble c-type cytochrome, designated c-556, was also isolated. The molecular mass, isoelectric point, spectrum, midpoint oxidation reduction potential and amino acid composition of this monoheam cytochrome are reported. The possible relationship of this cytochrome to other cytochromes c-556 is discussed. [ABSTRACT FROM AUTHOR]

The structure of a new antibiotic of the iturin group, bacillomycin F, has been demonstrated. It is a mixture of homologous peptidolipids, essentially C51H80N12O14 and C52N82N12O14. The lipid moiety consists of minor isoC15, anteisoC15 β-amino acids and major isoC16, isoC17 and anteisoC17 β-amino acids. The peptide sequence was determined by studying the peptides obtained after mild HCl hydrolysis and by Edman degradation of bacillomycin F treated with N-bromosuccinimide. The sequence was confirmed by two-dimensional NMR spectrometry and fast-atom-bombardment mass spectrometry gave the molecular masses of the homologous compounds. Bacillomycin F is a cyclic peptidolipid; its complete structure is given in the paper. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a heroin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3. Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion. [ABSTRACT FROM AUTHOR]

1. Leghemoglobin component a from Phaseolus vulgaris (kidney bean)was digested with trypsin; 15 tryptic peptides and free lysine were purified and the amino acid sequences of the peptides determined. 2. The internal order of the tryptic peptides was determined by the bridge peptides obtained from the thermolytic digest and the dilute acid hydrolyzate of kidney bean leghemoglobin α; 12 thermolytic peptides and two acid hydrolysis peptides were purified and the sequences were partially or completely determined. 3. The complete amino acid sequence of kidney bean leghemoglobin a is compared to that of leghemoglobin a from soybean (Glycine max) and to some animal globins. As regards sequence, the kidney bean globin has 79% identity with the soybean globin and 21 % identity with human hemoglobin γ-chain. Seven of the 14 amino acid residues common to most globins are found in the kidney bean globin. Trp-15 and Tyr-145 are evolutionarily conserved in this globin, which confirms the concept of a common origin of animal and plant globins. [ABSTRACT FROM AUTHOR]

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr. respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Osidatlon at 40°C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0°C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand. has a positive band at 340 nm, as m FAD. but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein. [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]

The mureins of several micrococi and coryneform organisms contain more than one mole of glutamic acid per mole of meso-diaminopimelic acid (Dpm). The amino acid sequence of these mureins was studied. The peptide subunit consists of Mur-L-Ala-γ-D-Glu-meso-Dpm-D-Ala, where Mur = muramic acid. The γ-carboxyl group of glutamic acid is substituted in most cases by a glycinc amide. The cross-linkage of the peptide subunits is performed by the dipeptide γ-D-glutamyl-D-glutamic acid. This peptide is bound by the γ-carboxyl group of the C-terminal glutamic acid to the amino group of diaminopimelic acid and to the carboxyl group of D-alanine of an adjacent peptide subunit by the amino group of the N-terminal glutamic acid. The mureins of the micrococci studied contain only one mole of amide ammonia per mole of diaminopimelic acid and this is bound to glycine. In the mureins of the coryneform organisms a second mole of amide ammonia was found which is probably bound to diaminopimelic acid. The murein of Staphylococcus lactis CCM 2432 lacks amide ammonia as well as glycine. [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]

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]

This paper describes a biochemical study on the protein composition of crystalline inclusions (cores) from plant peroxisomes. By SDS/PAGE and immunoblotting, a catalase of 59 kDa was identified as the predominant protein component in purified cores from sunflower (Helianthus annuus L.) cotyledons. A 55-kDa catalase was the only additional peptide detected. In contrast to in cores, the 55-kDa catalase was the major catalase protein in matrix fractions obtained from lysed peroxisomes. These findings suggested two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentation in sunflower cotyledons. Evidence for different amino acid sequences of the two catalases was found by peptide mapping with endoproteinase Glu-C, by expressing a eDNA encoding matrix catalase in Escherichia coli, and by partial amino acid sequencing of peptide fragments from 59-kDa core catalase. These results contradict the previous view that the formation of cores occurred via condensation of matrix catalase, and indicate that new concepts on the biogenesis and physiological function of plant peroxisomal cores need to be developed. [ABSTRACT FROM AUTHOR]

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism. [ABSTRACT FROM AUTHOR]

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

Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then adsorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide. [ABSTRACT FROM AUTHOR]

We have recently purified three distinct forms of fatty acid ω-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced eDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21665-21669]. The present paper describes cloning, sequencing and expression of a eDNA for the third fatty acid, ω-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the eDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled laurie acid was added to the culture medium of COS-7 cells transfected with the eDNA, significant amounts of radioactive dodecanedioic acid, together with ω- and (ω-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the ω- and (ω-1)-hydroxylation of laurie acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A. [ABSTRACT FROM AUTHOR]

This paper describes the elucidation of the primary structure of the three genetic variants of goat αs1-casein, αs1-Cn D, E and F, which have been found to be associated with reduced amounts of αs1-casein in milk. Variant E has the same electrophoretic mobility as variant B, but differs from the latter by the substitutions of Arg for Lys and of Thr for Ala at positions 100 and 195. A genetically controlled event which does not affect the amino acid sequence of this variant might be responsible for its lower rate of synthesis compared to that of αs1-casein B. The deletion of 11 amino acids at positions 59- 69 and of 37 amino acids at positions 59- 95 in variant B leads to variants D and F. In both cases the deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein. On the basis of sequence data for casein genes and cDNAs, it was concluded that the deletions occurring in the D and F variants are due to the exclusion of one and several exons, respectively. The observed deletions in the proteins could thus be the consequence of splice site mutations which would induce altered RNA processing and hence reduce the rate of synthesis of the casein. [ABSTRACT FROM AUTHOR]

From neurohaemal lobes of corpora cardiaca of Locusta migratoria a 5-kDa peptide has been isolated and its sequence established [see the accompanying paper, by Hietter et al. (1990) Eur. J. Biochem. 187, 241–247]. We have designed oligonucleotide probes from the peptide sequence of this molecule and screened a library prepared from mRNA of the neurosecretory cell region of the brain of this insect. Several positive cDNAs were isolated, the combined nucleotide sequences of which predict a large precursor of 145 residues (15770 Da) containing the newly isolated 5-kDa peptide. The peptide is flanked by regions homologous to the A and B chains of the superfamily of insulins. The overall organization of the precursor is as follows: signal peptide/domain homologous to the B chain of insulins/C (connecting)-peptide (corresponding to the newly isolated 5-kDa peptide)/domain homologous to the A chain of insulins. The numbers and relative positions of the cysteines of the Locusta peptide are equivalent to those of the other members of the insulin superfamily and most of the hydrophobic core residues are conserved. [ABSTRACT FROM AUTHOR]

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin. [ABSTRACT FROM AUTHOR]

We present in this paper the nucleotidic sequence of the FUR4 gene encoding the uracil permease in the yeast Saccharomyces cerevisiac. The deduced amino acid sequence of the permease has 633 residues; it consists of many hydrophobic stretches, only the N-terminal and C-terminal ends of the protein (about 100 and 50 amino acids respectively) being mostly hydrophilic. No N-terminal hydrophobic signal peptide is present, although it is shown in this work that the biosynthesis of the uracil permease goes through the secretion/glycosylation pathway. Using the results of three different methods, allowing the prediction of transmembrane α helices in proteic sequences, we drew a model of folding of the permease in the membrane. [ABSTRACT FROM AUTHOR]

High-molecular-mass protein was isolated from chicken heart muscle. The apparent molecular mass of a single polypeptide chain is similar to that of chicken gizzard filamin: 250–270 kDa. The protein interacts with antibodies against chicken gizzard filamin and induces F-actin gelation in a concentration-dependent manner. Immunofluorescent staining of cardiomyocytes and chicken heart sections with antifilamin antibody demonstrates two types of filamin localization: (a) filamin was located on the sarcomere border in the periphery of the Z-disk; (b) filamin was found in intercalated disks between cardiomyocytes. [ABSTRACT FROM AUTHOR]

Thyroglobulin, the dimeric glycoprotein (19 S, 2 × 330 kDa), specific to the thyroid gland, is the support for thyroid hormone synthesis. Elucidution of the mechanism for thyroid hormone synthesis requires the knowledge of the primary sequence of the protein. In this paper the sequence of the first coding 2190 nucleotides from the 5′ end of the human mRNA is presented. This was obtained by sequencing two previously described overlapping clones and by construction and sequencing of a single-stranded cDNA corresponding to the 5′ end of the mRNA. The nucleotide sequence represents a quarter of the human thyroglobulin mRNA, from which a polypeptide sequence of 730 amino acids at the NH2-terminal end of the monomer has been deduced. This sequence shows u repetition of five highly conserved motifs each of approximately 50 amino acids, the analysis of which allowed us to establish a consensus sequence. We have also demonstrated (a) the hormonogenic tyrosine residue recently described in the mature protein, which is located four amino acids alter the NH2-terminal ASn: (b) a prepeptide signal of thyroglobulin secretion comprising 19 amino acids preceding the Asn residue, the NH2-terminal residue of the mature protein and (c) a six-signal tripeptide (Asn-Xaa-Thr or Ser) of N-glycosylation of the chain. [ABSTRACT FROM AUTHOR]

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion, vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-1acking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast Rproteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities. [ABSTRACT FROM AUTHOR]

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and/easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1′ of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed m position P1. Position P2" accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3′ and P4′ were also defined by pool sequencing. Furthermore. pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library, and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase. [ABSTRACT FROM AUTHOR]

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

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

Flavocytochrome b2 from baker's yeast is a bifunctional tetramenc protein which carries two prosthetic groups, FMN and heme, per subunit of Mt, 58000. The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome b2 core (Mr 11000), homologous to cytochrome b5. It has been shown in the past that a number of proteases (yeast proteases, chymotrypsin) preferentially cleave the peptide chain at a point situated much further down the polypeptide chain than the C terminus of the hemeebinding domain, Some enzymatic parameters are concomitantly modified, but not the quaternary structure. This paper describes the conditions for selective proteolysis of intact flavocytochrome b2 and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8. Successive attack by a combination of two proteases is also described. We have established the amino acid sequence of the area where proteolytic attack takes places, and shown that chymotrypsin and S. aureus protease open only one bond, whereas yeast proteases remove five residues from the central part. The various nicked forms, some of which have lost up to 16 amino acid residues, have been enzymatically characterized. These and previous results lend support to, but do not prove, the idea that the flavodehydrogenase part of flavocytochrome b2 may be composed of two domains, linked by the region accessible to proteases. That area might constitute a hinge or rather a clasp between the domains. [ABSTRACT FROM AUTHOR]

Describes a nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. Backbone proteins of beta-sheet secondary structures; Hydrogen bond link of polypeptide strands; Amino acid chain resonance.

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of mouse pancreatic ribonuclease has been determined by analysis of tryptic, chymotryptic, thermolytic and CNBr peptides and by automatic sequence analysis of the intact protein. The sequence of mouse RNase differs in 20–30% of the positions from other RNase sequences. Three unique or nearly unique substitutions were found, viz. Gly-68 → Arg-68, Arg-85 → His-85 and Ser-123 → Thr-123. All these three residues might be involved in interactions with substrate molecules. A most parsimonious tree of the myomorph rodent RNase shows that after the divergence of rat and mouse, the ribonuclease of rat accumulated substitutions at a rate 2.5–4.3 times as high as the rates in other branches of the tree and 23 times as high as the average rate in the Bovidae ribonuclease evolution. These extreme fluctuations in substitution rate are difficult to reconcile with the hypothesis of the evolutionary clock. The high evolution rate of rat ribonuclease is thought to be caused by positive selection, leading to new functional properties of the enzyme. [ABSTRACT FROM AUTHOR]

Earlier papers from our laboratory reported the nucleotide sequence of the beginning of the simian virus 40 HindII + III fragment D (Hind-D, which contains the information for the presumed initiation codon for the VP2 protein), HindII + III fragment E (Hind-E), and HindII + III fragment K (Hind-K, which contains the information for the termination signal for the VP2 and VP3 proteins and the initiation signal for the VP1 protein). We report here the nucleotide sequence of the remainder of fragment Hind-D as determined by the partial, chemical degradation procedure of Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. (1977) 74, 560–564]. As the aforementioned restriction fragments cover the total of the overlapping VP2 and VP3 genes, the entire nucleotide sequence of these genes, as well as the derived amino acid sequence of the VP2 and VP3 proteins, is now known. Assuming that eukaryotic protein synthesis always starts with an AUG codon (and not with a GUG) and considering various evidence about the position of the VP3 gene within the VP2 gene, we conclude that the second AUG codon in phase coded by fragment Hind-D serves as the initiation codon for the translation of VP3. The first part of VP2 (98 residues), which is not present in VP3, is very hydrophobic and contains no cysteine or basic amino acids; possibly it serves for binding to, or penetrating through, membranes. The very basic carboxyl terminus of both VP2 and VP3 may possibly be involved in attachment to the DNA. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [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 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 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 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]