Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Complete amino acid sequence of the major component myoglobin of finback whale (Balaenoptera physalus)

The complete amino acid sequence of the major component myoglobin from finback whale, Balaenoptera physalus, was determined by the automated Edman degradation of several large peptides obtained by specific cleavages of the protein. Three easily separable peptides were obtained by cleaving with cyanogen bromide at the two methionine residues and one large peptide was isolated after cleavage with (2-p-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. More than 60% of the covalent structure was established by the sequential degradation of three of these peptides and the apomyoglobin. An additional 30% of the primary sequence was established with peptides obtained from tryptic digestion of both the apomyoglobin and the acetimidoapomyoglobin, and the final 10% of the sequence was completed after digestion of the two larger cyanogen bromide peptides with S. aureus strain V8 protease. This myoglobin differs from that of the sperm whale, Physeter catodon, at 15 positions, from that of the arctic minke whale, Balaenoptera acutorostrata, at 3 positions, and from that of the California gray whale, Eschrichtius gibbosus, at 4 positions. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).     

Characterization of three peptides derived from prosomatostatin [prosomatostatin-(1-63)-, -(65-76)- and -(79-92)-peptides] in a human pancreatic tumour.

By using only reverse-phase h.p.l.c., three fragments of prosomatostatin were isolated from an extract of a human pancreatic neuroendocrine tumour that produced somatostatin, vasoactive intestinal polypeptide and gastrin-releasing peptide. The amino acid composition of the peptides indicated that they represented prosomatostatin-(1-63)-peptide, prosomatostain-(65-76)-peptide and prosomatostatin-(79-92)-peptide (somatostatin-14). The identity of prosomatostatin-(1-63)-peptide was confirmed by characterization of the products of digestion with Armillaria mellea (honey fungus) proteinase. Partial micro-sequencing of prosomatostatin-(1-63)-peptide showed that the Gly24-Ala25 bond of preprosomatostatin was the site of cleavage of the signal peptide. Thus human prosomatostatin is a protein of 92 amino acid residues that is proteolytically cleaved in a pancreatic tumour at the site of a dibasic-residue (arginine-lysine) processing site and at a single-monobasic-residue (arginine) processing site.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena.

1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Studies on the covalent structure of eland pancreatic ribonuclease.

Studies on the covalent structure of eland (Taurotragus oryx) pancreatic ribonuclease have been performed on tryptic and thermolysin digests. The first 45 residues have been determined with a Beckman sequencer. From the remaining part of the sequence only those peptides were sequenced that differed in amino acid composition with the corresponding peptide of bovine ribonuclease. Eland pancreatic ribonuclease differs in four positions from bovine pancreatic ribonuclease A, but more differences due to a different state of amidation may be present. The absence of an Asn-X-Thr/Ser sequence in the covalent structure of eland ribonuclease (asparagine 34 has been substituted by aspartic acid) explains the absence of a glycosidated component in eland ribonuclease.     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

Comparison of the primary structures of ribonuclease U2 isoforms.

The primary structures of the two isoforms of ribonuclease U2, RNAases U2-A and U2-B, were analysed and compared with each other. Among the chymotryptic peptides obtained from the reduced and S-carboxymethylated enzymes, only peptides C-3 were different from each other in terms of chromatographic behaviour on reverse-phase h.p.l.c. On the basis of chemical analyses of these peptides, it was shown that RNAase U2-B had an isopeptide bond in which Asp-32 was linked to Gly-33 through the beta-carboxy group in its side chain instead of the alpha-carboxy group. Deamidation of Asn-32 in RNAase U2-A led to the formation of this unusual linkage. The previously reported sequence of RNAase U2 [Sato & Uchida (1975) Biochem. J. 145, 353-360] was corrected by changing amino acid residues at eight different positions and by inserting an asparagine residue at position 32. The numbering of the positions of amino acid residues located downstream of Asn-32 was therefore shifted by 1. Accordingly, RNAase U2-A was shown to be composed of 114 amino acid residues.     

The primary structure of a PYY-related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY.

Although the amino acid sequence of members of the pancreatic polypeptide (PP)-family of regulatory peptides has been poorly conserved during vertebrate evolution, the overall length of the peptides (36 amino acid residues) has remained constant. Nucleotide sequence analysis of cloned cDNAs and/or genomic fragments has shown the PP-related sequence immediately follows the signal peptide in the prepropeptides. A peptide tyrosine-tyrosine (PYY)-related peptide with 37 residues has been isolated from the chicken intestine, and its primary structure was established as: Ala-Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly10-Asp-Ala-Ala-Ser-P ro-Glu-Glu-Ile-Ala-Gln20-Tyr-Phe-Ser-Ala-Leu-Arg-His-Tyr-Il e-Asn30-Leu-Val-Thr-Arg-Gln-Arg-Tyr.CONH2. The presence of an additional alanine residue at the NH2-terminus of the peptide suggests that the site of cleavage of the signal peptide in chicken preproPYY is different from the site of cleavage in other PP-family prepropeptides.     

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.     

Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76)

A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.     

Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile.

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.