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.

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 Preliminary characterization of the multiple forms of ram sperm hyaluronidase

1988 ◽  
Vol 252 (3) ◽  
pp. 875-882 ◽  
Author(s):  
R A Harrison
Keyword(s):  
Vinyl Alcohol ◽  
Specific Activity ◽  
Antigenic Determinants ◽  
Poly Vinyl Alcohol ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Low Concentrations ◽  
Poly Ethylene ◽  
Ram Sperm ◽  
Oligomeric Series

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.

Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle

1988 ◽  
Vol 66 (10) ◽  
pp. 1023-1031 ◽  
Author(s):  
Peter A. Nagainis ◽  
Frederick H. Wolfe ◽  
Shridhar K. Sathe ◽  
Darrel E. Goll
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Maximal Activity ◽  
Chicken Breast ◽  
Ph Optimum ◽  
Tissue Extracts ◽  
Chicken Skeletal Muscle

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90–95% complete after 10 s at 0 °C. Autolysis for 15 s at 0 °C converts the proteinase from a form requiring 250–300 μM Ca2+ to one requiring 9–10 μM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immedately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.

Studies on the toxicity of copper. I. The toxic action of copper in vivo and in vitro

1966 ◽  
Vol 166 (1004) ◽  
pp. 273-284 ◽  
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Membrane Surface ◽  
High Specific Activity ◽  
Rapid Onset ◽  
Significant Inhibition ◽  
Low Concentrations ◽  
The Brain

With the object of throwing light upon the brain damage found in patients with Wilson’s disease (hepato-lenticular degeneration) due to the accumulation of copper, the effect of Cu 2+ has been investigated in pigeons. Subarachnoid injections of Cu 2+ (10 to 25 µ g) led to rapid onset of convulsions and death. These concentrations of Cu 2+ inhibited pigeon and rat b rain mitochondria; more organized tissue breis or slices showed no significant inhibition of oxygen up take at Cu +2 concentration inducing convulsions in vivo . Studies with radioactive copper ( 64 Cu) showed that the injected copper was widely distributed in the brain, though maximal near the site of injection. Centrifugation showed a high specific activity in the ATP -ase-rich microsomal fraction. Thorium in concentrations similar to Cu 2+ was not toxic. From this we suggest that the Cu 2+ does not alter the charge on some membrane surface. Since the effect of the copper is immediate, and since it does not affect respiration of slices in these low concentrations, we conclude that it is exerting its convulsive effect directly upon the cell surfaces.

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 Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

1989 ◽  
Vol 262 (2) ◽  
pp. 409-416 ◽  
Author(s):  
G A Saravani ◽  
D A Cowan ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Serine Proteinase ◽  
Gel Permeation Chromatography ◽  
Exchange Chromatography ◽  
Acetate Buffer ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Low Ionic Strength

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

scholarly journals Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase

1976 ◽  
Vol 154 (2) ◽  
pp. 257-264 ◽  
Author(s):  
H H Tai ◽  
C L Tai ◽  
C S Hollander
Keyword(s):  
Enzyme Activity ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Enzyme System ◽  
Prostaglandin F2α ◽  
Thiol Group ◽  
Rabbit Kidney ◽  
Prostaglandin D2 ◽  
Kidney Medulla ◽  
Prostaglandin Synthase

A simple radioactive-substrate assay for prostaglandin synthase (EC 1.14.99.1), which uses t.l.c. to measure simultaneously different prostaglandins synthesized from one precursor substrate, was developed. Rabbit kidney-medulla prostaglandin synthase catalyses the formation of prostaglandin E2, prostaglandin F2α and prostaglandin D2 from arachidonic acid. Fractionation of crude homogenates indicated that the microsomal fraction possessed the highest specific activity of prostaglandin synthase, whereas the soluble fraction exhibited little enzyme activity but rather contained a heat-labile inhibitory macromolecular factor(s), which might be attributed to the serum albumin present in this fraction. The microsomal fraction possessed low intrinsic enzyme activity, but the actvity could be fully stimulated by the presence of both GSH (reduced glutathione) and a phenolic cofactor. Only cysteine could partially replace GSH, whereas other thiols were inactive and some were even inhibitory. A variety of phenolic compounds, including catecholamines, dopamine (3,4-dihydroxyphenethylamine), 5-hydroxytryptamine and quinol, were active in stimulating prostaglandin synthase. In all cases, the stimulation was reflected in the synthesis of all three prostaglandins with ratios not significantly altered by different phenolic cofactors. The synthesis of each of the different prostaglandins appeared to have similar pH optima. The enzyme system was not inhibited by thiol-group inhibitors or a variety of metal chelators except for cyanide and 8-hydroxyquinoline. Characterization of the kidney-medulla prostaglandin synthase system indicated that it exhibited properties similar to those of the enzyme system present in seminal vesicles.

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 Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase

1979 ◽  
Vol 177 (1) ◽  
pp. 283-288 ◽  
Author(s):  
H G Nimmo
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Heat Inactivation ◽  
Mitochondrial Enzyme ◽  
Microsomal Enzyme ◽  
Glycerol Phosphate ◽  
Low Concentrations ◽  
High Concentrations

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.

scholarly journals A shift in the optimum pH of phospholipase D produced by activating long-chain anions

1969 ◽  
Vol 112 (5) ◽  
pp. 795-799 ◽  
Author(s):  
R. H. Quarles ◽  
R. M. C. Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Activity Profile ◽  
Ph Optimum ◽  
Surface Ph ◽  
Electrophoretic Mobilities ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Long Chain

1. The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) towards ultrasonically treated phosphatidylcholine or large phosphatidylcholine particles activated with ether was maximal near pH5, and there was little activity above pH6. 2. When the enzyme was activated by the addition of phosphatidic acid to large phosphatidylcholine particles the pH optimum was shifted to pH6·5 irrespective of the amount of activator added. 3. When the enzyme was activated with low concentrations of dodecyl sulphate the pH optimum was 5·5 with little activity above pH6. With higher concentrations of dodecyl sulphate the pH–activity profile was shifted upwards towards a pH optimum of 6·5–6·6, the magnitude of the shift depending on the extent of the hydrolysis. 4. The shifts in the pH–activity profiles cannot be correlated with changes in the ‘surface pH’ of the substrate particles calculated from the measurement of their ζ-potentials (electrophoretic mobilities).

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