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.

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 Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme

1979 ◽  
Vol 177 (2) ◽  
pp. 649-659 ◽  
Author(s):  
R C Davies ◽  
A Neuberger
Keyword(s):  
High Activity ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Accurate Determination ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synthetase Activity ◽  
Activity Form ◽  
Rhodopseudomonas Spheroides

1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000–170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000–68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer, pH 7.4. 6. Enzyme samples possessed an intrinsic yellow fluorescence when viewed under u.v. light and this fluorescence coincided exactly with enzymic activity on gel electrophoresis. Fluorescence maxima were 420 nm (excitation) and 495 nm (emission). 7. Radioactive 35S-labelled enzyme had 14 atoms of sulphur/mol of protein (or/40 leucine residues) of which 5–6 residues were cyst(e)ine and 8–9 residues were methionine. 8. Mo carbohydrate was detected apart from glucose, which prevented accurate determination of tryptophan with methanesulphonic acid and tryptamine.

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.

Localization of carbonic anhydrase in the cytoplasmic membrane of Neisseria sicca (strain 19)

1981 ◽  
Vol 27 (1) ◽  
pp. 87-92 ◽  
Author(s):  
M. N. MacLeod ◽  
I. W. DeVoe
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Carbonic Anhydrase Activity ◽  
Protein Patterns ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Neisseria Sicca

The carbonic anhydrase activity and the growth of Neisseria sicca 19 were inhibited by the sulfonamide acetazolamide (10−5 M). Such inhibition was completely overcome by the addition of exogenous bicarbonate. Some carbonic anhydrase activity associated with the membranous envelope fraction of the cell was released when cells were broken by sonic treatment but not during cell breakage by high-pressure extrusion. After the selective solubilization (4 °C) of the inner membrane of envelopes by treatment with 1% sodium lauroyl sarcosinate, all detectable carbonic anhydrase activity was found in the soluble (inner membrane) fraction. After fractionation of the cell envelope into inner and outer membranes by treatment with ethylenediaminetetraacetate (EDTA) followed by sucrose density gradient centrifugation, the total and specific activity of carbonic anhydrase paralleled that of succinate dehydrogenase, an inner membrane enzyme marker. The Coomassie blue stained protein patterns after polyacrylamide gel electrophoresis of the bands from the sucrose density gradient provided confirmation that the inner and outer membranes had indeed been separated.

scholarly journals Carbonic Anhydrase III Is Not Required in the Mouse for Normal Growth, Development, and Life Span

2004 ◽  
Vol 24 (22) ◽  
pp. 9942-9947 ◽  
Author(s):  
Geumsoo Kim ◽  
Tae-Hoon Lee ◽  
Petra Wetzel ◽  
Cornelia Geers ◽  
Mary Ann Robinson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Carbon Dioxide ◽  
Carbonic Anhydrase ◽  
Physiological Function ◽  
Specific Activity ◽  
Normal Growth ◽  
Standard Laboratory ◽  
Wild Type ◽  
Carbonic Anhydrase Iii ◽  
Growth Development

ABSTRACT Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Purified Factor XII Has a Higher Specific Activity than the Parent Molecule in Plasma

1991 ◽  
Vol 65 (02) ◽  
pp. 169-173 ◽  
Author(s):  
Walter A Wuillemin ◽  
Miha Furlan ◽  
Isabella Huber ◽  
Bernhard Lämmle
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xii ◽  
Parent Molecule ◽  
Proteolytic Activation ◽  
Filtration Step

SummaryThe specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/ mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII.In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.

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.

CuZn superoxide dismutase activities from Fasciola hepatica

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 555-562 ◽  
Author(s):  
L. PIACENZA ◽  
R. RADI ◽  
F. GOÑI ◽  
C. CARMONA
Keyword(s):  
Molecular Weight ◽  
Superoxide Dismutase ◽  
Fasciola Hepatica ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Soluble Extract ◽  
Sod Activity

The levels of superoxide dismutase (SOD) were determined in detergent-soluble, somatic and excretion–secretion (E–S) preparations from adult Fasciola hepatica using the xanthine oxidase system and visualized in substrate gels. Compared to detergent-soluble and somatic extracts, E–S products showed the highest SOD activity (88 ·5 U/mg), indicating active release to the medium in which parasites were maintained. SOD specific activity was also detected at high levels in E–S products from 3-week-old and 5-week-old immature migrating flukes (25 and 143 U/mg, respectively). In all preparations except for the somatic extract, the activity was characterized as cyanide-sensitive CuZn SOD. Differences in SOD isoenzyme profiles between the extracts were observed in native polyacrylamide gel electrophoresis: the somatic and detergent-soluble extracts exhibited 1 band of activity while the E–S products from immature and adults flukes contained 2 and 3 migrating bands, respectively. SOD was purified from the detergent-soluble extract and E–S products of adult worms by a combination of ultrafiltration, gel filtration on Sephacryl S-200 HR and ion-exchange chromatography on QAE Sephadex A-50. The SOD from detergent-soluble extract showed, by SDS–PAGE analysis, 1 band of 16 kDa apparent molecular weight. The SOD from E–S products showed 2 bands of 16 and 60 kDa apparent molecular weight. N-terminal sequence analysis of the 16 kDa band from the detergent-soluble preparation showed some similarity with Schistosoma mansoni cytoplasmic SOD. These enzymes may have a potential role in the evasion of the oxidative burst killing mechanism by immune cells.

Purification and Properties of a Porcine Tissue Plasminogen Activator and its Comparison with Activators from Human Uterine Tissue, Blood and Urine

1979 ◽  
Author(s):  
P. Kok
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
Uterine Tissue ◽  
Porcine Tissue ◽  
Swedish Medical ◽  
Isoelectric Ph

A plasminogen activator was prepared from porcine ovaries by a simple procedure described earlier (Kok and Astrup, Biochemistry 8:79, 1969) and further purified by ion exchange chromatography on SE- or SP-Sepharose and affinity chromatography on Sepharose-lysine.The preparation was homogeneous by gel electrophoresis at pH 3.5 and SDS-polyacrylamide electrophoresis(PAGE). The specific activity was about 4.5 x 106 A and A tissue activator units per mg protein (one tissue activator unit is comparable to 0.1 CTA urokinase unit in a fibrinolytic assay under specified conditions). The molecular size estimated by SDSPAGE corresponded to a Mw of 67000 dalton. After reduction with dithiothreitol the activator produced a single band corresponding to a Mw of 33500 dalton, indicating cleavage into two chains of equal size. Isoelectric focusing revealed several bands, which were active and with an isoelectric pH range of 7-8.An activator from human uterine tissue and an activator in the euglobulin fraction from human plasma were similar to the porcine tissue activator in molecular size, estimated by gel filtration, and immunochemically. The porcine tissue activator differed from the activator from human urine in reactivity, molecular size and immunochemically.(This work was supported by USPHS Grant HL-05020, NIH, National Heart and Lung Institute, to dr. Tage Astrup and by the Swedish Medical Research Council (project no. 13X 03906 07A).)

scholarly journals Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate

1987 ◽  
Vol 246 (2) ◽  
pp. 263-269 ◽  
Author(s):  
P J Casey ◽  
J M Lowenstein
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Rat Skeletal Muscle ◽  
Adenylosuccinate Lyase ◽  
Apparent Homogeneity ◽  
High Concentrations

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at −70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

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