scholarly journals α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Williams ◽  
H Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

scholarly journals Purification and properties of an α-d-galactoside galactohydrolase from the seeds of Trifolium repens (white clover)

1977 ◽  
Vol 161 (3) ◽  
pp. 509-515 ◽  
Author(s):  
J Williams ◽  
H Villarroya ◽  
F Petek
Keyword(s):  
White Clover ◽  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Alpha Galactosidase

Five alpha-D-galactosidases (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) have been identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidase I has been purified to homogeneity with an approx. 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the alpha-D-galactosidic activity. The pH optimum was found to be between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans has been examined, and it has been found that alpha-galactosidase I exhibits two enzymic activities, namely alpha-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick & Smith (1968), and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the mol.wt. has been estimated to be 43 000 and 41 000 respectively. These results indicate that alpha-galactosidase I is a monomeric protein and that both enzymic activities associated with the enzyme reside on the same polypeptide chain.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

scholarly journals Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani

1977 ◽  
Vol 167 (3) ◽  
pp. 685-692 ◽  
Author(s):  
David B. Harper
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Fusarium Solani ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sole Source ◽  
Polyacrylamide Gel ◽  
Ph Optimum

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH4)2SO4 precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a Km with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.

scholarly journals Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain

1980 ◽  
Vol 187 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Lilian M. Y. Lee ◽  
Antonieta K. Salvatore ◽  
Peter R. Flanagan ◽  
Gordon G. Forstner
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J.173, 553–563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3–5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

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