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.

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.

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.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits

1982 ◽  
Vol 60 (4) ◽  
pp. 463-470 ◽  
Author(s):  
T. Youdale ◽  
J. P. MacManus ◽  
J. F. Whitfield
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Purification And Properties ◽  
Exclusion Chromatography

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

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 The purification and properties of the second component of guinea-pig complement

1982 ◽  
Vol 205 (1) ◽  
pp. 59-67 ◽  
Author(s):  
M A Kerr ◽  
J Gagnon
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Sequence Homology ◽  
Polypeptide Chain ◽  
Classical Pathway ◽  
Factor B ◽  
Single Polypeptide Chain ◽  
Purification And Properties ◽  
Rate Of Decay ◽  
Single Polypeptide

A method has been developed for the purification to homogeneity of guinea-pig complement component C2. Contrary to previous reports, guinea-pig C2 is a single polypeptide chain with apparent mol.wt. of 102000, the same as human C2. It is cleaved by C1s to yield fragments C2a (apparent molwt. 74000) and C2b (apparent mol.wt. 34000). The amino acid composition and N-terminal sequences of these fragments are similar to those of human C2a and C2b. Human and guinea-pig C2 show more extensive sequence homology to Factor B than previously identified. The known homology around the sites of cleavage by C1s and Factor D has now been extended by a stretch of ten identical or conservatively substituted residues. Sequence homology has now been identified at the N-terminal of C2b and Factor Ba. The properties of the classical-pathway C3 convertases assembled from human C4b, C1s and human or guinea-pig C2 have been compared. The rates of cleavage of human and guinea-pig C2 by C1s (and therefore the rates of assembly of the C3 convertases) are similar. The rate of decay of the activity of the C3 convertase formed from guinea-pig C2 is 10-fold lower than for human C2. This greater stability reflects a higher affinity of guinea-pig C2a for human C4b. The presence of C2b is not necessary for C3 convertase activity.

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.

Adipocyte insulin-binding species: the 40 Å Stoke's radius protein

1984 ◽  
Vol 62 (7) ◽  
pp. 566-570
Author(s):  
C. Elliott ◽  
H. Joseph Goren
Keyword(s):  
Insulin Receptor ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Relative Mass ◽  
Single Polypeptide Chain ◽  
Dodecyl Sulfate ◽  
Single Polypeptide ◽  
Adipocyte Plasma Membranes

Several laboratories have demonstrated the presence of large (70 Å) (1 Å = 0.1 nm) and small (40 Å) insulin receptors. This report provides evidence that the 40 Å insulin receptor migrates on dodecyl sulfate – acrylamide gel electrophoresis as a 90 000 dalton protein and that this protein is a single polypeptide chain. 125I-labeled insulin was bound to plasma membranes from isolated rat adipocytes. Following removal of unbound 125I-labeled insulin, the mixture was exposed to disuccinimidyl suberate. Proteins tagged with 125I-labeled insulin were separated by dodecyl sulfate gel electrophoresis or Sepharose 6B chromatography. Autoradiography of the gels demonstrated several large (relative mass (Mr) > 300 000) and one small (Mr ~ 90 000) labeled protein in nonreduced membrane proteins. Dithiothreitol reduction decreased the large insulin-binding species to its known subunits, but the 90 000 dalton protein did not decrease in size. Triton X-100 solubilized plasma membranes were separated by Sepharose 6B chromatography. One labeled protein, with Kav = 0.57 elution position, on dodecyl sulfate gel electrophoresis migrated as a 90 000 dalton protein. Thus, rat adipocyte plasma membranes contain both an oligomeric insulin-binding species and a monomeric insulin-binding species. The relationship of the monomeric to the oligomeric insulin receptor is discussed.

Xylose metabolism in Pachysolen tannophilus: purification and properties of xylose reductase

1984 ◽  
Vol 30 (11) ◽  
pp. 1330-1336 ◽  
Author(s):  
Günther Ditzelmüller ◽  
Christian P. Kubicek ◽  
Wilfried Wöhrer ◽  
Max Röhr
Keyword(s):  
Xylose Reductase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Xylose Metabolism ◽  
Pachysolen Tannophilus ◽  
Single Polypeptide Chain ◽  
Purification And Properties ◽  
Single Polypeptide ◽  
Reduction Charge

Xylose reductase (xylitol: NADP oxidoreductase, EC 1.1.1.139) has been purified from D-xylose grown cells of the yeast Pachysolen tannophilus by application of DEAE-cellulose ion exchange chromatography, 2′,5′-ADP-Sepharose affinity chromatography, Biogel P200 gel filtration, and dextran blue Sepharose chromatography to approximately 95% homogeneity. It consists of a single polypeptide chain with a relative molecular weight of 35 000–40 000 and an isoelectric point of pH 4.9. The enzyme has a broad substrate specificity similar to that of aldose (or aldehyde) reductases from mammalian tissues. It exhibits Michaelis–Menten type kinetics (Km D-xylose, 162 mM; Km D-xylitol, 212 mM; Km NADPH, 0.059 mM; [Formula: see text], 0.071 mM). The enzyme is specific for NADPH; activity with NADH is below 0.5% of Vmax observed with NADPH. The reduction of xylose is inhibited by NADP, the anabolic reduction charge (NADPH/NADP + NADPH), and also in a complex manner by ATP. At physiological pH values the equilibrium is Keq = 10−10. The importance of these findings for the physiology of xylose fermentation by this yeast is discussed.

scholarly journals Cyclization Reaction Catalyzed by Glycogen Debranching Enzyme (EC 2.4.1.25/EC 3.2.1.33) and Its Potential for Cycloamylose Production

2002 ◽  
Vol 68 (9) ◽  
pp. 4233-4239 ◽  
Author(s):  
Michiyo Yanase ◽  
Hiroki Takata ◽  
Takeshi Takaha ◽  
Takashi Kuriki ◽  
Steven M. Smith ◽  
...  
Keyword(s):  
High Performance ◽  
Anion Exchange Chromatography ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Minimum Size ◽  
Rapid Reduction ◽  
Debranching Enzyme ◽  
Single Polypeptide Chain ◽  
Glycogen Debranching Enzyme ◽  
Single Polypeptide

ABSTRACT Glycogen debranching enzyme (GDE) has 4-α-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type α-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic α-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-α-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.

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.

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