Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)

1984 ◽  
Vol 62 (8) ◽  
pp. 699-708 ◽  
Author(s):  
K. Shamsuzzaman ◽  
N. F. Haard
Keyword(s):  
Gastric Mucosa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Porcine Pepsin ◽  
Milk Clotting

Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS) – polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogenous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS–polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2–3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.

Isolation and characterization of cathepsin D from human gastric mucosa

1981 ◽  
Vol 46 (13) ◽  
pp. 3302-3313 ◽  
Author(s):  
Jan Pohl ◽  
Ladislav Bureš ◽  
Karel Slavík
Keyword(s):  
Gastric Mucosa ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Human Gastric Mucosa ◽  
Ph Optimum ◽  
Isolation And Characterization

The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

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 cis, cis-Muconate cyclase from Trichosporon cutaneum

1980 ◽  
Vol 191 (1) ◽  
pp. 37-43 ◽  
Author(s):  
A Gaal ◽  
H Y Neujahr
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Trichosporon Cutaneum ◽  
Thiol Groups ◽  
Divalent Metal Ions ◽  
Ph Optimum

The inducible enzyme catalysing the conversion of cis, cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis, cis-muconate, but not with cis, trans- or trans, trans-muconate or 3-carboxy-cis, cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis, cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis, cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of 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 Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae

1998 ◽  
Vol 64 (10) ◽  
pp. 3607-3614 ◽  
Author(s):  
Christine Riou ◽  
Jean-Michel Salmon ◽  
Marie-Jose Vallier ◽  
Ziya Günata ◽  
Pierre Barre
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Glucose Tolerant

ABSTRACT Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1mg of protein−1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki , 1.36 M) or glucono-δ-lactone (Ki , 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

scholarly journals Purification and properties of a specific collagenase from rabbit synovial fibroblasts

1975 ◽  
Vol 151 (3) ◽  
pp. 645-653 ◽  
Author(s):  
Z Werb ◽  
J J Reynolds
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Synovial Fibroblasts ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Purification And Properties ◽  
Fibroblast Collagenase ◽  
Specific Rabbit

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35°C the purified enzyme could hydrolyse >50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24°or 35°C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35°C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.

Photoactivated linkage of catechol O-methyltransferase and S-adenosyl-L-methionine

1984 ◽  
Vol 62 (10) ◽  
pp. 964-969 ◽  
Author(s):  
Peter H. Yu
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Ultraviolet Light ◽  
Paper Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Comt Inhibitors ◽  
Isoelectric Focussing

The formation of a stably linked complex of tritiated S-adenosyl-L-methionine (AdoMet) and catechol O-methyltransferase (COMT) has been achieved by irradiating the enzyme and ligand in Tris–HCl buffer (pH 7.5) with ultraviolet light at 254 nm. The reaction is specific as shown by a number of criteria. COMT inhibitors such as S-adenosylhomocysteine can block this photoactivated linkage. The [3H]AdoMet–COMT adduct has been shown to be a homogeneous protein by Sephadex gel filtration, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and isoelectric focussing. After extensive proteolysis of the [3H]AdoMet–COMT adduct with pronase P, one major labelled product was released. This fragment could be separated by paper chromatography and was shown to be chromatographically identical to that released from the [3H]AdoMet – phenylethanolamine N-methyltransferase adduct.

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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