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.

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.

Partial purification of a hypertensive substance from rat erythrocytes

1985 ◽  
Vol 63 (10) ◽  
pp. 1321-1326 ◽  
Author(s):  
William D. McCumbee ◽  
Gary L. Wright
Keyword(s):  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Calcium Uptake ◽  
Specific Activity ◽  
Present Report ◽  
Fold Increase ◽  
Partial Purification ◽  
Rat Erythrocytes ◽  
In Vitro Effects

A crude extract prepared from rat erythrocytes was previously shown to contain an active component (hypertensive factor, HF) that stimulates the in vitro uptake of calcium by aortic rings and causes a sustained and severe elevation in systolic blood pressure in normotensive rats. The present report demonstrates that the in vitro effects of HF on calcium uptake are concentration dependent and reversible, and that these effects on calcium uptake provide a convenient bioassay for monitoring the purification of HF. HF was prepared from a diffusate obtained by dialyzing hemolyzed rat erythrocytes. The diffusate was applied successively to molecular sieve and anion exchange columns resulting in a 1500-fold increase in specific activity relative to the starting hemolysate. The compound stimulating calcium uptake was found to be heat stable and resistant to digestion with trypsin and chymotrypsin. In contrast, treatment with pronase E, a nonspecific protease, markedly increased the calcium transport stimulatory activity. The administration of the purified preparation to normotensive rats having a systolic blood pressure of 118 ± 2 Torr (1 Torr = 133.322 Pa) resulted in a significant elevation of blood pressure (171 ± 11 Torr) by day 5. The severity of this increase further suggests that there is a marked enhancement in potency relative to earlier fractions that were examined. Perhaps most importantly, these results indicate that, at this stage of purification, the pressor component and the calcium-stimulatory component of HF appear to co-purify.

scholarly journals Partial purification and some properties of a cholinesterase from bush bean (Phaseolus vulgaris L.) roots

1978 ◽  
Vol 175 (3) ◽  
pp. 769-777 ◽  
Author(s):  
D H Mansfield ◽  
G Webb ◽  
D G Clark ◽  
I E P Taylor
Keyword(s):  
Phaseolus Vulgaris ◽  
Specific Activity ◽  
Apparent Molecular Weight ◽  
Fold Increase ◽  
Partial Purification ◽  
Phaseolus Vulgaris L ◽  
Native Form ◽  
Bush Bean ◽  
Electric Eel ◽  
Stokes Radius

A cholinesterase was partially purified from bush bean (Phaseolus vulgaris L.) roots by using acridinium-based ligand affinity chromatography. The procedure gave a 78-fold increase in specific activity, although at least three inactive contaminants remained. The enzyme activity was maximal against acetyl esters of choline and was inhibited by neostigmine. Di-isopropyl phosphorofluoridate completely inhibited activity at concentrations greater than 0.1 mM. The catalytic centre activity was 2 × 10(-4) times that of electric eel acetylcholinesterase. Cholinesterase activity appeared as a peak (s = 4.2 +/- 0.1 S) after isokinetic sedimentation. The Stokes radius was 4.00 nm and the apparent molecular weight was 72700 +/- 1900. The smallest active and native form of the enzyme appeared to be a monomer. This contrasts with animal acetylcholinesterases, in which the smallest active and native forms are multimeric.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

scholarly journals Effect of hepatic injury on prolyl 3-hydroxylase and 4-hydroxylase activities in rat liver and on immunoreactive prolyl 4-hydroxylase concentrations in the liver and serum

1978 ◽  
Vol 170 (1) ◽  
pp. 129-135 ◽  
Author(s):  
J Risteli ◽  
L Tuderman ◽  
K Tryggvason ◽  
K I Kivirikko
Keyword(s):  
Protein Concentration ◽  
Hepatic Injury ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Partial Purification ◽  
Hydroxylase Activity ◽  
Enzyme Protein ◽  
Carbon Tetrachloride Administration ◽  
Total Enzyme

After severe hepatic injury induced by dimethylnitrosamine, approximately a 4-fold increase in hepatic prolyl 4-hydroxylase activity occurred within 4 days, whereas the increases in total immunoreactive prolyl 4-hydroxylase protein and in prolyl 3-hydroxylase activity were only about 1.4-fold. The different magnitudes of the increases in the prolyl 4-hydroxylase and 3-hydroxylase activities were verified after partial purification of the enzymes by gel filtration. The data support previous reports indicating differential increases in the activities of individual enzymes of collagen biosynthesis in hepatic injury. Separation of prolyl 4-hydroxylase tetramers from the monomer-size protein by gel filtration indicated that the increase in enzyme activity was similar to that in enzyme tetramers, and an increase had also occurred in the ratio of enzyme tetramers to total enzyme protein. Thus the specific activity of the tetramers had remained unchanged in liver injury. The administration of dimethylnitrosamine was also accompanied by a marked increase in the immunoreactive prolyl 4-hydroxylase protein concentration in the serum, and a similar effect was also noted after carbon tetrachloride administration, results suggesting that the increases originated in the liver.

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 Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system

1975 ◽  
Vol 146 (3) ◽  
pp. 713-722 ◽  
Author(s):  
K P Wheeler ◽  
J A Walker ◽  
D M Barker
Keyword(s):  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Total Phospholipid ◽  
Kidney Tissue ◽  
Residual Activity ◽  
Fold Increase ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Phospholipid Content ◽  
Total Phospholipid Content

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.

scholarly journals A calcium ion-dependent adenosine triphosphate pyrophosphohydrolase in plasma membrane from rat liver. Demonstration that the adenosine triphosphate analogues adenosine 5′-[βγ-imido]triphosphate and adenosine 5′-[βγ-methylene]-triphosphate are substrates for the enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 817-820 ◽  
Author(s):  
H Flodgaard ◽  
C Torp-Pedersen
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphate ◽  
Rat Liver ◽  
Specific Activity ◽  
Fold Increase ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Phosphate Bond ◽  
Hydrolysis Of ◽  
Fold Activation

An ATP pyrophosphohydrolase in a rat liver plasma-membrane subfraction was studied with respect to specific Ca2+ activation of the beta-phosphate bond hydrolysis. ATP and, in addition, adenosine 5′-[betagamma-imido]triphosphate and adenosine 5′-[betagamma-methlylene]triphosphate were substrates for Ca2+-stimulated enzymic hydrolysis of the beta-phosphate bond. A 15-fold activation was observed by raising the free Ca2+ concentration from 10(-7) to 10(-5) M. Mg2+ had little effect. Solubilization in 1% deoxycholate and partial purification on a sucrose density gradient resulted in a 5-fold increase in specific activity with unaltered Ca2+-stimulation pattern. The possible importance of the enzyme in Ca2+ transport is discussed.

scholarly journals The differential release of basal ATPase, Ca2+-dependent ATPase, 5′-nucleotidase and cholesterol during homogenization of skeletal muscle

1980 ◽  
Vol 188 (2) ◽  
pp. 569-572 ◽  
Author(s):  
E J Barrett ◽  
N M Ryan ◽  
D R Headon
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fragmentation Pattern ◽  
Specific Concentration ◽  
Dependent Atpase

The influence of homogenization times on the presence of constituents in the microsomal fraction of skeletal muscle was investigated. Membranes having Ca2+-activated ATPase activity have a fragmentation pattern distinct from that of membranes displaying Ca2+-independent or basal ATPase activity. These latter membranes were found in highest specific concentration in the microsomal fraction prepared from homogenates subjected to short periods of homogenization. 5′-Nucleotidase (EC 3.1.3.5) activity paralleled that of basal ATPase on short periods of homogenization, as also did the specific concentration of cholesterol. Longer periods of homogenization led to a decrease in the specific activity of basal atpase, which reached its lowest value at 120s of homogenization, whereas the specific activity of 5′-nucleotidase and the specific concentration of cholesterol decreased initially in a similar manner to basal ATPase, but both increased substantially after the longest period of homogenization.

scholarly journals CYTOCHEMISTRY OF PHOSPHATASES OF THE SARCOPLASMIC RETICULUM

1966 ◽  
Vol 31 (3) ◽  
pp. 473-487 ◽  
Author(s):  
A. G. Engel ◽  
Lois W. Tice
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Psoas Muscle ◽  
Thiol Group ◽  
Ph Optimum ◽  
Dependent Atpase ◽  
Synergistic Activation ◽  
Low Concentrations ◽  
Ouabain Inhibition

A microsomal fraction was isolated from rabbit psoas muscle by a modification of Muscatello's method. The fraction contained a Mg-dependent ATPase which had a pH optimum of 7.5. Activity was further stimulated by addition of Na or K or other monovalent cations to the reaction mixture, but synergistic activation by Na and K, and ouabain inhibition, could not be demonstrated. The enzyme hydrolyzed only ATP (adenosine triphosphate) and ITP (inosine triphosphate) at appreciable rates, but Na or K stimulated activity only when ATP was used as substrate. Activity was inhibited by Ca and by low concentrations of Na deoxycholate, and was sensitive to inhibition by thiol group reagents. The enzyme could be distinguished from another enzyme, also present in the fraction, which was Ca-activated, and which exhibited a wider substrate specificity, different pH activation characteristics, lower specific activity, lack of stimulation by Na or K, and less sensitivity to inhibition by deoxycholate and by thiol group reagents. These findings formed the basis for demonstration of the Mg-dependent ATPase in situ.

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