The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum, is identical to cytochrome C-oxidase, peptide VII

The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum is reported. The molecule is known to rapidly alter the proportions of digestive enzymes secreted by the rabbit pancreas in vivo and in vitro, by selection of the specific intra-pancreatic source from which the preset mixture of digestive enzymes is secreted. The sequence is identical to that of cytochrome C-oxidase peptide VII (cCoVII) from bovine heart, with the exception of a substitution of threonine for alanine at position 6 and a second substitution of alanine for threonine at position 71. Disulfide bridges link positions 29-64 and 39-53. cCoVII-chymodenin has a pentapeptide (-Ala-Glu-Gly-Thr-Phe-) near the carboxy-terminus which is immediately preceded by an -Arg-Arg- sequence in the porcine and bovine sequences of cCoVII. This peptide is identical to a pentapeptide found close to the amino terminus of the hormones gastric inhibitory peptide (GIP) and glucagon-like peptide I. The identity to cCoVII means chymodenin as isolated is itself unlikely to be a gastrointestinal hormone. However, the partial commonality of sequence with the glucagon-secretin family immediately adjacent to a pro-hormone-like activation site, and the specific actions on the exocrine pancreas, means that the molecule probably mimics the natural actions of an as-yet uncharacterized member of the glucagon family, which exerts a unique action on exocrine pancreatic secretion.     

Acidic phospholipases A2 from the venom of common sea snake enhydrina schistosa

Two acidic phospholipases A have been purified from the venom of common sea snake (Enhydrina schistosa) by chromatography on Sephadex G-75 gel media, Bio-Rex 70 ion-exchanger followed by repeated Chromatography on DEAE-Sephadex A-25 ion-exchanger. The two preparations were shown to be homogeneous by polyacrylamide gel electrophoresis and ion-exchange chromatography. The enzymes were shown to be specific for the ‘two’ position of egg yolk lecithin. The molecular weight of both enzymes determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was approx. 14000. Both enzymes were non-lethal. Amino acid composition data indicated high contents of aspartic acid, glycine and alamine in both enzymes.     

The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids.

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 x 10(6)-5 x 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.     

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.     

Chemical Cleavage of Bovine β-Lactoglobulin by BNPS-Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine β-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

Purification to apparent homogeneity of a thymocyte specific growth factor from calf thymus

A thymic peptide previously found to recruit thymocytes from G1 into S phase has been purified from a crude thymic extract by subsequent steps of gel exclusion chromatography and reverse phase high performance liquid chromatography (HPLC). The purified material, which appeared homogeneous on thin-layer chromatography and HPLC, stimulated the DNA synthesis of cultured guinea pig thymocytes in a nanomolar concentration range. The amino acid composition revealed a high content of acidic amino acids and no apparent homology to previously defined growth factors and thymus differentiation hormones.     

Confirmation of direct angiotensin formation by kallikrein.

This study was undertaken to confirm our previous preliminary observation that hog pancreas kallikrein (EC 3.4.21.35) directly liberated an angiotensin-like substance from human plasma protein Cohn fraction IV-4 at an acidic pH of 4.0-5.0. First, the possibility of proangiotensin or des-Asp1-angiotensin being the pressor substance was ruled out by t.l.c. Secondly, the pressor substance was purified by Sephadex G-25 and Bio-Gel P-2 gel filtration, and finally by high-performance liquid chromatography. The amino acid composition of the isolated pressor substance (residues/mol) was: Asp, 1.03; Val, 1.03; Ile, 1.00; Tyr, 0.69; Phe, 1.04; His, 0.91; Arg, 0.86; Pro, 0.86. This composition was identical with that of angiotensin. Since the reaction mixture was not contaminated with common proteolytic enzymes, such as trypsin, chymotrypsin, renin, cathepsin D and proangiotensin-converting enzyme, and other enzymes activated by kallikrein, it is clear that hog kallikrein directly produces angiotensin in vitro.     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

Purification of a lysinonorleucine cross-linked peptide fraction from porcine aorta elastin.

Highly purified elastin from porcine aorta was submitted to elastase digestion. The enzymic products were subjected to gel filtration on Sephadex G 25. The excluded fraction was then submitted to thermolysin digestion and gel filtration. The excluded fraction was submitted to partial acid hydrolysis and gel filtration. Several sub-fractions were obtained. The F3 subfraction containing cross-linking agents (desmosines and lysinonorleucine) was finally subjected to ion exchange chromatography. A highly enriched peptide fraction containing lysinomorleucine was obtained and then purified by preparative electrophoresis. The ratio of enrichment passed from 2 residues of lysinonorleucine (expressed as lysine) from starting material (elastin) to 178 out of 1,000 residues in the final step. In this peptide fraction, if we express in molar ratio and consider the amount of lysinonorleucine is one residue, the following amino-acid composition is Pro:3, Gly:1, Ala:2, LNL:1, Lys:2. No traces of desmosines are detected. The role of lysinonorleucine in bridging functions in elastin is discussed.     

Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1

Serum-free culture medium conditioned by an established human pancreatic adenocarcinoma cell line, CAPAN-1, contains copious amounts of immunoreactivity due to pancreatic secretory trypsin inhibitor (PSTI) as demonstrated by radioimmunoassay. The immunoreactive substance was purified from the conditioned medium to apparent homogeneity by trypsin affinity and gel filtration chromatography with an overall recovery of 40%, and its primary structure was determined by Edman degradation. The immunoreactive substance is a peptide of 56 amino acid residues with a calculated molecular weight of 6,241. Its amino acid composition, primary structure, and inhibitory effect against trypsin are indistinguishable from those of human pancreatic juice PSTI, indicating that this substance is PSTI itself. This is the first direct demonstration that tumor cells secrete PSTI in vitro. When CAPAN-1 was inoculated into a nude mouse, it produced a tumor and the tumor synthesized human PSTI in vivo, as demonstrated by the fact that the tumor extract contained 99.0 +/- 26.2 ng of human PSTI/mg of protein, while PSTI was not detected in extracts from other tissues examined. Furthermore, high levels of human PSTI (14.3 +/- 2.6 ng/ml) were detected in the serum of tumor-bearing mice but not in that of nontumor-bearing mice, suggesting that PSTI secreted from the tumor appears in the blood circulation. Taken together, these results strongly support the view that the serum levels of PSTI are elevated in cancer-bearing patients due to secretion of this peptide from tumor cells per se.     

Isolation and partial chemical characterization of THF, a thymus hormone involved in immune maturation of lymphoid cells.

A procedure for isolation and characterization of the active principle of calf thymus is described. It consists of homogenization, ultracentrifugation and dialysis of the material. The active dialyzate is further purified by gel filtration on Sephadex G-10 and G-25 columns followed by anion exchange chromatography. The level of purification is assessed by isoelectric focusing on polyacrylamide gels. The active principle is a polypeptide of MW 3220. Determination of the amino acid composition revealed the presence of high proportion of acidic residues.     

Purification and properties of bacteriolytic enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans

To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37 degrees C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.     

Characterization of Potent Fibrinolytic Enzymes in Earthworm,Lumbricus rubellus

Stable and potent fibrinolytic enzymes (six homogeneous proteins) were purified to homogeneity from extracts of the lyophilized powder of an earthworm, Lumbricus rubellus. The molecular weight of each enzyme estimated by SDS–polyacrylamide gel electrophoresis was different from those by gel filtration chromatography in the six purified proteins. The exact molecular weight of each enzyme (F-III-2, F-III-1, F-II, F-I-2, F-I-l, and F-I-0) measured by ionspray MS analysis was 29, 662, 29, 667, 24, 664, 24, 220, 24, 196, and 23,013, respectively. The isoelectric point (pI) of each enzyme was 3.40, 3.60, 4.20, 4.00, 4.30, and 4.85, respectively. The enzymes were single polypeptide chains. They had a very strong fibrinolytic activity and the maximum reactivity for chromogenic substrates from pH 9-11. The enzymes, acidic proteins that had abundant asparagine and aspartic acid, and low lysine in their amino acid composition, did not contain component sugars. The enzymes were stable at from pH 1-11 and up to 60°C. Studies on substrate specificity and inhibition indicated that these enzymes were alkaline trypsin-like serine proteases. N-Terminal amino acid sequences of the enzymes had local similarities to those of trypsin-like enzymes such as elastase and coagulation factor IX. From the results of amino acid sequence, amino acid composition analyses and immunological analyses, it was suggested that these six enzyme proteins were derived as isozyme(s) from at least four different genes.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

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.     

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Chemical cleavage of bovine beta-lactoglobulin by BNPS-skatole for preparative purposes: comparative study of hydrolytic procedures and peptide characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine -lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

NADH: (acceptor) oxidoreductase from bovine adrenal medulla chromaffin granules

The NADH:(acceptor) oxidoreductase from membranes of bovine adrenal medulla chromaffin granules has been purified by column chromatography. After solubilization of the membranes with emulphogen, a nonionic detergent, the enzyme was purified by dye-ligand chromatography and gel filtration. The oxidoreductase appeared essentially homogeneous on two gel electrophoretic systems. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme revealed a dimeric structure with a combined molecular weight of about 55,000. The enzyme eluted as a detergent-lipid-protein aggregate with a Stoke's radius of 43 Å on gel filtration columns in the presence of emulphogen. The amino acid composition of the oxidoreductase was found to be distinct from that of similar enzymes from other organelles. Topographical experiments indicated that the enzyme is a transmembrane protein.     

Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)