scholarly journals A novel proteinase from human pancreas

1984 ◽  
Vol 219 (3) ◽  
pp. 735-742 ◽  
Author(s):  
A Sziegoleit
Keyword(s):  
Binding Protein ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Human Pancreas ◽  
Single Polypeptide Chain ◽  
Quantitative Measurements ◽  
Alpha 1 Antitrypsin ◽  
Single Polypeptide ◽  
Nitrophenyl Ester ◽  
Cholesterol Binding

A cholesterol-binding protein was previously isolated from human pancreas [Sziegoleit (1982) Biochem. J. 207, 573-582] and shown to consist of a single polypeptide chain with an apparent Mr of 28 000 and an isoelectric point of pH 4.9. In further investigations, a proteolytic activity was observed to be present in preparations of this protein. The enzyme activity was not dissociable from the cholesterol-binding protein. It decreased in the presence of sodium dodecyl sulphate or urea parallel to degradation of the protein, indicating autodegradation in the presence of these denaturants. Glucagon digestion studies indicated the carbonyl bond of alanine to be a favoured site of the enzymic cleavage. The proteinase was inactive against chromogenic substrates relatively specific for elastase, trypsin and chymotrypsin, but was found to cleave benzyloxycarbonylalanine p-nitrophenyl ester efficiently. The enzyme was inactivated by phenylmethanesulphonyl fluoride and was thus classified as a serine proteinase. Autoradiographic studies demonstrated binding to serum alpha 1-antitrypsin and alpha 2-macroglobulin in a similar manner to that observed with other pancreatic endo-proteinases. The collective results indicate that the isolated protein, provisionally named ‘cholesterol-binding pancreatic proteinase’, is a novel proteinase of the human pancreas. Quantitative measurements indicate that it comprises 4-6% of total protein in pancreatic secretions.

scholarly journals Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells

1983 ◽  
Vol 212 (1) ◽  
pp. 219-222 ◽  
Author(s):  
M Kondo ◽  
Y Koshihara ◽  
M Kawamura ◽  
S Murota
Keyword(s):  
Polypeptide Chain ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Prostaglandin Endoperoxide ◽  
Single Polypeptide Chain ◽  
Free Translation ◽  
Prostaglandin Endoperoxide Synthase ◽  
Single Polypeptide

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.

scholarly journals Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides

1985 ◽  
Vol 225 (1) ◽  
pp. 135-142 ◽  
Author(s):  
J Gagnon ◽  
G J Arlaud
Keyword(s):  
Serine Proteinase ◽  
Complete Sequence ◽  
Classical Pathway ◽  
Human Complement ◽  
Chain Fragment ◽  
Single Polypeptide Chain ◽  
Tryptic Cleavage ◽  
Cnbr Cleavage ◽  
Single Polypeptide ◽  
A Chain

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.

scholarly journals Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(ββ)

1999 ◽  
Vol 12 (5) ◽  
pp. 395-405 ◽  
Author(s):  
Takanori Tanaka ◽  
Naofumi Miwa ◽  
Satoru Kawamura ◽  
Hitoshi Sohma ◽  
Katsutoshi Nitta ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Calcium Binding ◽  
Polypeptide Chain ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Single Polypeptide Chain ◽  
Single Polypeptide

scholarly journals Purification and characterization of a cholesterol-binding protein from human pancreas

1982 ◽  
Vol 207 (3) ◽  
pp. 573-582 ◽  
Author(s):  
A Sziegoleit
Keyword(s):  
Serum Proteins ◽  
Binding Capacity ◽  
Binding Protein ◽  
Protein Composition ◽  
Sodium Deoxycholate ◽  
Gel Chromatography ◽  
Human Pancreas ◽  
Lipid Complex ◽  
Intestinal Fluids ◽  
Cholesterol Binding

The protein composition of human intestinal lavage fluids was analysed by electroimmunoassay. In addition to secretory immunoglobulin A and other components that were antigenically related to serum proteins, a number of gut-specific proteins were detected. One of these was found to exhibit the capacity of binding sodium deoxycholate and cholesterol. After isolation of this cholesterol-binding protein from intestinal fluids, immunohistochemical studies utilizing a specific antiserum indicated the pancreas to be the organ of its synthesis. The protein was subsequently purified from necrobiotic pancreas tissues and was found to be composed of a single polypeptide chain with a mol. wt. of 28 000 and an isoelectric point of pH4.9. The deoxycholate binding capacity determined by gel chromatography in the presence of [3H]deoxycholate was calculated to be approx. 24 mol of deoxycholate/mol of protein. In the intestinal fluids the protein appeared to be present in firm association with cholesterol, phospholipids, triacylglycerols and bile salts as a macromolecular protein-lipid complex. The possibility is raised that the pancreas-derived, cholesterol-binding protein may fulfil a function as an intestinal ‘lipoprotein’.

scholarly journals Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion

1979 ◽  
Vol 179 (3) ◽  
pp. 593-602 ◽  
Author(s):  
W J Gullick ◽  
D G Herries ◽  
E J Wood
Keyword(s):  
Lymnaea Stagnalis ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Weight Fraction ◽  
Single Polypeptide Chain ◽  
Domain Structures ◽  
Single Polypeptide

The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 × 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the ‘collar’ of the molecule, and an aggregate some 20–30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000–300,000.

Human placental β-galactosidase: structural and immunological observations

1984 ◽  
Vol 62 (6) ◽  
pp. 529-534 ◽  
Author(s):  
Christopher S. Jones ◽  
Donald Mahuran ◽  
J. Alexander Lowden ◽  
John W. Callahan
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Relative Mass ◽  
Antibody Preparation ◽  
Single Polypeptide Chain ◽  
Apparent Homogeneity ◽  
Activity Measurements ◽  
Single Polypeptide ◽  
Simultaneous Loss

β-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of β-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr β-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with β-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate – polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against β-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of β-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

The yeast aminopeptidase Y

1988 ◽  
Vol 34 (2) ◽  
pp. 118-124 ◽  
Author(s):  
J. Nowak ◽  
H. Tsai
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Imino Group ◽  
Peptide Bonds ◽  
Single Polypeptide Chain ◽  
Peptidase Inhibitor ◽  
Terminal Amino ◽  
Single Polypeptide ◽  
Hydrolysis Of ◽  
Serine Endopeptidases

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate – polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S—S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free α-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X—Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp—X and Glu—X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40–50% glycerol, at −20 °C.

The purification and properties of (1→4)-β-D-glucan cellobiohydrolase from Sclerotium rolfsii: substrate specificity and mode of action

1984 ◽  
Vol 62 (9) ◽  
pp. 920-926 ◽  
Author(s):  
Rajkumar V. Patil ◽  
Jai C. Sadana
Keyword(s):  
Sodium Dodecyl ◽  
Sclerotium Rolfsii ◽  
Degree Of Polymerization ◽  
Relative Mass ◽  
Cellulolytic Enzyme ◽  
Transferase Activity ◽  
Total Carbohydrate ◽  
Single Polypeptide Chain ◽  
Purification And Properties ◽  
Single Polypeptide

A cellulolytic enzyme which shows high activity towards H3PO4 – swollen cellulose, but no viscosity-lowering activity towards carboxymethylcellulose, has been purified from the culture filtrates of Sclerotium rolfsii. The purified enzyme is homogeneous in disc gel electrophoresis, with and without sodium dodecyl sulfate, and in analytical isoelectric focusing in polyacrylamide gel. The enzyme is a glycoprotein containing 7.0% total carbohydrate and has a relative mass of 41 500 and an isoelectric point of 4.32. The enzyme is composed of a single polypeptide chain and has no cystine or half-cystine residues. It is specific for β-D-(1→4)-linkage. The principal product from each of the substrate was cellobiose (93–96%); glucose was also detected (4–7%). The rate of cellodextrin hydrolysis increased as the degree of polymerization increased. The enzyme has been identified as (1→4)-β-D-glucan cellobiohydrolase. The enzyme does not show transglycosylase or transferase activity.

scholarly journals A 33 kDa serine proteinase from Scedosporium apiospermum

1996 ◽  
Vol 315 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Gérald LARCHER ◽  
Bernard CIMON ◽  
Franç;oise SYMOENS ◽  
Guy TRONCHIN ◽  
Dominique CHABASSE ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Biochemical Properties ◽  
Enzyme Synthesis ◽  
Scedosporium Apiospermum ◽  
Human Fibrinogen ◽  
Single Polypeptide Chain ◽  
Single Polypeptide

An extracellular proteinase produced by the filamentous fungus Scedosporium apiospermum has been purified and characterized. Initially, in vitro conditions for enzyme synthesis were investigated. The highest yield of enzyme production was obtained when the fungus was cultivated in modified Czapek–Dox liquid medium supplemented with 0.1% bacteriological peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the proteinase was accomplished by (NH4)2SO4 precipitation, followed by gel filtration through Sephadex G-75 and finally affinity chromatography through immobilized phenylalanine. Analysis of the purified enzyme by SDS/PAGE revealed a single polypeptide chain with an apparent molecular mass of 33 kDa. Further investigation of its physical and biochemical properties disclosed numerous similarities with those of the previously described serine proteinase of Aspergillus fumigatus. The enzyme was not glycosylated and its pI was 9.3. Proteinase activity was optimum between 37 and 50 °C and at pH 9.0, but remained high within a large range of pH values between 7 and 11. The inhibition profile and N-terminal amino acid sequencing confirmed that this enzyme belongs to the subtilisin family of serine proteinases. In agreement with this, the specific synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to be an excellent substrate for the proteinase, with an estimated Km of 0.35 mM. Like the alkaline proteinase of A. fumigatus, this enzyme was able to degrade human fibrinogen, and thus may act as a mediator of the severe chronic bronchopulmonary inflammation from which cystic fibrosis patients suffer.

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