scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

scholarly journals Carbonic anhydrase isoenzymes in the erythrocytes and uterus of the rabbit

1970 ◽  
Vol 120 (2) ◽  
pp. 299-310 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbon Dioxide ◽  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Zinc Content ◽  
Exchange Chromatography ◽  
Uterine Tissue ◽  
Marked Rise ◽  
Activity Form

1. Two forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from rabbit erythrocytes and two forms from rabbit uterine tissue (endometrium) in the progestational stage of pregnancy (days 6–8 of gestation). Separation of the isoenzymes was achieved by ion-exchange chromatography, preparative polyacrylamide-gel electrophoresis and isoelectric focusing. A comparison was made of the general properties and kinetic behaviour of the purified isoenzymes. 2. Although indistinguishable in terms of molecular weight and zinc content the isoenzymes were very different as catalysts of the hydration of carbon dioxide. The two erythrocyte isoenzymes, found in almost equal amounts, differed more than 100-fold in specific activity. Of the two isoenzymes prepared from either endometrial or entire uterine homogenates one was kinetically indistinguishable from the erythrocyte high-activity form, whereas the other, also possessing high activity, was found only in the endometrial or uterine tissue. Present evidence suggests that the former isoenzyme originated from residual blood contaminating the tissue homogenates, and that a marked rise in the content of the latter isoenzyme accounts for the increase in rabbit endometrial carbonic anhydrase activity that previously has been observed in early pregnancy. 3. Minor forms of the erythrocyte isoenzymes, having a characteristic quantitative and electrophoretic relationship to one another, were occasionally produced during purification. 4. The actions were investigated of the inhibitors acetazolamide (5-acetamido-3,4-diazole-1-thia-2-sulphonamide), 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxyzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was superior to the high-activity form as a catalyst of β-naphthyl acetate hydrolysis.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

scholarly journals The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

1970 ◽  
Vol 120 (4) ◽  
pp. 797-808 ◽  
Author(s):  
M. J. Carter ◽  
D. S. Parsons
Keyword(s):  
Gastrointestinal Tract ◽  
Carbonic Anhydrase ◽  
Guinea Pig ◽  
High Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Carbonic Anhydrases ◽  
Low Activity

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (`high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (`low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). `High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly `low activity' carbonic anhydrases from those tissues were indistinguishable. `High activity' carbonic anhydrase was markedly different from the `low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

1988 ◽  
Vol 250 (2) ◽  
pp. 453-458 ◽  
Author(s):  
H Sobek ◽  
H Görisch
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Acidocaldarius ◽  
Sedimentation Equilibrium ◽  
Prolonged Storage ◽  
Heat Stable ◽  
Equilibrium Data ◽  
Poly Ethylene ◽  
Hydrolysis Of

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.

scholarly journals Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 174 ◽  
Author(s):  
Manami Kobayashi ◽  
Yuya Kumagai ◽  
Yohei Yamamoto ◽  
Hajime Yasui ◽  
Hideki Kishimura
Keyword(s):  
Main Product ◽  
Specific Activity ◽  
Red Alga ◽  
Glycoside Hydrolase Family ◽  
Bifidobacterium Adolescentis ◽  
Glycoside Hydrolase Family 43 ◽  
Key Enzyme ◽  
Hydrolysis Of ◽  
Low Activity ◽  
Hydrolase Family

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

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