scholarly journals The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate

1979 ◽  
Vol 179 (2) ◽  
pp. 431-438 ◽  
Author(s):  
M V Bell ◽  
J R Sargent
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Anguilla Anguilla ◽  
Chloride Cells ◽  
Partial Purification ◽  
Sodium Orthovanadate ◽  
Turnover Number ◽  
Ouabain Binding ◽  
Beta Adrenergic Agonists

1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572–4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.

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 Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification

1983 ◽  
Vol 214 (1) ◽  
pp. 69-75 ◽  
Author(s):  
P B Moore ◽  
N Kraus-Friedmann
Keyword(s):  
Affinity Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fold Increase ◽  
Partial Purification ◽  
Dependent Atpase

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.

A light microscopic study of the action of sodium orthovanadate on the gills of fresh-water and sea-water eels (Anguilla anguilla L.)

1979 ◽  
Vol 59 (4) ◽  
pp. 859-865 ◽  
Author(s):  
K. F. Kelly ◽  
B. J. S. Pirie ◽  
M. V. Bell ◽  
J. R. Sargent
Keyword(s):  
Light Microscopy ◽  
Fresh Water ◽  
Constant Pressure ◽  
Sea Water ◽  
Anguilla Anguilla ◽  
Chloride Cells ◽  
Sodium Orthovanadate ◽  
Central Venous ◽  
Water Fish ◽  
Gill Filaments

Gills of fresh-water and sea-water eels were perfused at a constant pressure with physiological Ringer containing 10−6 M sodium orthovanadate and examined by light microscopy. The secondary gill filaments were markedly vasoconstricted in both freshwater and sea-water fish although the peripheral blood route around the secondary filaments was unaffected. The central venous space in the primary filament was largely unaffected. Significant constriction of both afferent and efferent arteries on the primary filament occurred. We conclude that orthovanadate vasoconstricts eel gills mainly at the level of the secondary filaments. The study also emphasizes that chloride cells are located on both the primary and secondary filaments of fresh-water gills but solely on the primary filaments of sea-water gills.

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

2019 ◽  
Vol 24 (7) ◽  
pp. 59
Author(s):  
Reemy M. Mohamed Saleh ◽  
Firas T . Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Polycystic Ovary Syndrome ◽  
Specific Activity ◽  
Polycystic Ovary ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Ovary Syndrome ◽  
Electrophoresis Method ◽  
High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206).    http://dx.doi.org/10.25130/tjps.24.2019.130

Partial purification and characterization of a soluble acid phosphatase from the tapeworm, Hymenolepis diminuta

1991 ◽  
Vol 65 (2) ◽  
pp. 103-110
Author(s):  
Michael J. Bumbulis ◽  
Peter W. Pappas
Keyword(s):  
Acid Phosphatase ◽  
Cellular Localization ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Hymenolepis Diminuta ◽  
Partial Purification ◽  
Transferase Activity ◽  
Crude Enzyme Preparation ◽  
Ammonium Sulphate Precipitation ◽  
And Function

ABSTRACTAn acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130 000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44°C and pH 5·0. The enzyme appeared to be a monomer with a molecular weight of approximately 62 000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

scholarly journals Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens

1982 ◽  
Vol 203 (1) ◽  
pp. 209-221 ◽  
Author(s):  
G Murphy ◽  
J J Reynolds ◽  
U Bretz ◽  
M Baggiolini
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Polymorphonuclear Leucocyte ◽  
Partial Purification ◽  
Type I ◽  
Polymorphonuclear Leucocytes ◽  
Gelatinase Activity ◽  
Apparent Homogeneity

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases (‘TIMP’) and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.

scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

scholarly journals The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (Anguilla anguilla) adapted to fresh water and sea water

1974 ◽  
Vol 144 (1) ◽  
pp. 69-75 ◽  
Author(s):  
John R. Sargent ◽  
Alison J. Thomson
Keyword(s):  
Fresh Water ◽  
Adenosine Triphosphatase ◽  
Sea Water ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Anguilla Anguilla ◽  
Gill Tissue ◽  
Sodium Deoxycholate ◽  
Enzyme Preparations ◽  
Protein Components

1. Gill tissue from eels adapted to fresh water or to sea water was disrupted in 0.32m-sucrose containing 0.1% (w/v) sodium deoxycholate and the subcellular distribution of (Na++K+)-dependent adenosine triphosphatase was determined. 2. About 70% of the recovered enzyme was in a fraction sedimenting between 225000gav.-min and 6000000gav.-min; the specific activities of enzymes from tissues of freshwater and seawater eels were 16 and 51 μmol of phosphate/h per mg of protein respectively. 3. The enzymes from gills of freshwater and seawater eels were indistinguishable on the basis of a number of parameters. These included phosphorylation by [γ-32P]ATP, the binding of [3H]ouabain, the extent to which bound [3H]ouabain was displaced by increasing concentrations of KCl and pH optima. 4. Electrophoresis on polyacrylamide gels in sodium dodecyl sulphate showed that enzyme preparations from both sources had an identical number of protein components. 5. The higher specific activity of (Na++K+)-dependent adenosine triphosphatase from tissue of seawater eels was accompanied by increased amounts of two protein components. One of these proteins retained 32P after treatment of the enzyme with [γ-32P]ATP and had mol.wt. 97000; the other component was a glycoprotein with mol.wt. approx. 46000. 6. The results are discussed in terms of the nature of the transepithelial NaCl pumps in the gills of freshwater and seawater fish.

Proton-translocating ATPase from bovine kidney medulla: partial purification and reconstitution

1988 ◽  
Vol 254 (1) ◽  
pp. F71-F79 ◽  
Author(s):  
S. Gluck ◽  
J. Caldwell
Keyword(s):  
Proton Transport ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Intact Protein ◽  
Partial Purification ◽  
Kidney Medulla ◽  
Bovine Kidney ◽  
Vacuolar Acidification ◽  
Proton Translocating Atpase

The proton-translocating ATPase that is responsible both for urinary and vacuolar acidification was partially purified from bovine kidney medulla microsomes. ATPase activity was purified to a maximum specific activity of 1.7 mumol.min-1.mg prot-1 and was inhibited completely by N-ethylmaleimide. The relative molecular weight (Mr) of the intact protein estimated by high-pressure size-exclusion liquid chromatography was 586,000. Nondenaturing gels of the isolated enzyme revealed two protein bands at MrS of 551,000 and 523,000. Sodium dodecyl sulfate-gel electrophoresis of the isolated H+-ATPase revealed component subunits at MrS of 70,000, 56,000, 45,000, 42,000, 38,000, 31,000, 15,000, 14,000, and 12,000. The properties of the isolated H+-ATPase and of microsomal ATP-dependent proton transport correlated closely. The isolated H+-ATPase was reconstituted into phospholipid liposomes and demonstrated N-ethylmaleimide-inhibitable ATP-dependent potential generation, consistent with electrogenic proton transport. In overall structure, the enzyme appears to be a new type of H+-ATPase with several features of the F0F1 class of ion-translocating ATPases but is immunologically and structurally different from the mitochondrial F1-ATPase.

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