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 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.

DISTRIBUTION OF CHOLINESTERASE IN NORMAL HUMAN MUSCLE

1963 ◽  
Vol 41 (8) ◽  
pp. 1713-1720 ◽  
Author(s):  
J. Crispin Smith ◽  
Vera M. Foldes ◽  
Francis F. Foldes
Keyword(s):  
Skeletal Muscle ◽  
Microsomal Fraction ◽  
Cholinesterase Activity ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Human Muscle ◽  
Ventricular Muscle ◽  
Cardiac Ventricular Muscle ◽  
Normal Human ◽  
Quadriceps Femoris Muscles

The intracellular distribution of acetylcholinesterase and butyrylcholinesterase in biopsied human rectus abdominis and quadriceps femoris muscles was investigated. Differential ultracentrifugal fractionation showed that the highest specific activity of acetylcholinesterase was associated with the microsomal fraction, this enzyme being concentrated 19-fold compared to the original homogenate. The highest specific activity of butyrylcholinesterase was found in the non-particulate fraction. The mitochondrial fraction showed the lowest specific cholinesterase activity. Wide variation of cholinesterase activity was found in various autopsied muscles, the highest being oculomotor muscle and the lowest cardiac ventricular muscle. About 90% of the cholinesterase activity of skeletal muscle was due to acetylcholinesterase and 10% to butyrylcholinesterase.

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 effect of dantrolene on skeletal-muscle sarcoplasmic-reticulum function in malignant hyperpyrexia in pigs

1983 ◽  
Vol 212 (2) ◽  
pp. 399-405 ◽  
Author(s):  
M D White ◽  
J G Collins ◽  
M A Denborough
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Muscle Relaxant ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Malignant Hyperpyrexia ◽  
Dependent Atpase ◽  
Sarcoplasmic Reticulum Membrane

The effect of the muscle relaxant dantrolene on isolated sarcoplasmic reticulum was studied in control and malignant-hyperpyrexia-susceptible Landrace pigs. The membranes prepared from both sources showed similar Ca2+-dependent ATPase activities, had comparable phospholipid/protein ratios, and their sodium dodecyl sulphate/polyacrylamide-gel patterns were indistinguishable. Membranes from both sources appeared to bind similar amounts of dantrolene. The drug did not stimulate Ca2+-dependent ATPase activity in preparations from either source. The rates of Ca2+ exchange and Ca2+ efflux appeared to be similar in sarcoplasmic reticulum of control and malignant-hyperpyrexia-susceptible pigs. Dantrolene did not affect either the rates or the amount of Ca2+ lost from the vesicles. These results suggest that dantrolene does not directly affect the movement of Ca2+ across the sarcoplasmic-reticulum membrane.

Effect of calcium concentration on isolated hamster brain Na-K-, Mg-dependent ATPase

1982 ◽  
Vol 60 (8) ◽  
pp. 1119-1124 ◽  
Author(s):  
F. Rohani ◽  
J. D. Welty ◽  
D. F. Hastings
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Specific Activity ◽  
Hill Coefficient ◽  
Affinity Binding ◽  
Dependent Atpase ◽  
Positive Cooperativity

In these experiments the effect of different concentrations of calcium on the specific activity of isolated Na-K-ATPase was studied. The result of these investigations showed that calcium at 10−6 and 10−7 M stimulated the Na-K-ATPase activity. These studies also show that at higher calcium concentrations (10−5–10−3 M), the activity of the enzyme is inhibited. The results from calcium binding to isolated membranes, rich in Na-K-ATPasc, strongly suggest the existence of a low-affinity binding site which exhibits a large positive cooperativity, Kd = 2.8 × 10−5 ± 0.4 × 10−5 M and Hill coefficient of 2.9 ± 0.2. The calcium concentration (1.9 × 10−5 M sufficient to produce significant (24%) inhibition of the Na-K-ATPase is approximately equal to the Kd observed for calcium binding.

scholarly journals Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver

1976 ◽  
Vol 157 (3) ◽  
pp. 705-712 ◽  
Author(s):  
P V Sulakhe ◽  
S J Sulakhe ◽  
N L Leung ◽  
P J St Louis ◽  
R A Hickie
Keyword(s):  
Skeletal Muscle ◽  
Cerebral Cortex ◽  
Adenylate Cyclase ◽  
Cardiac Muscle ◽  
Guanylate Cyclase ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Frog Heart ◽  
Dependent Atpase ◽  
Absolute Requirement

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.

DISTRIBUTION OF CHOLINESTERASE IN NORMAL HUMAN MUSCLE

1963 ◽  
Vol 41 (1) ◽  
pp. 1713-1720 ◽  
Author(s):  
J. Crispin Smith ◽  
Vera M. Foldes ◽  
Francis F. Foldes
Keyword(s):  
Skeletal Muscle ◽  
Microsomal Fraction ◽  
Cholinesterase Activity ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Human Muscle ◽  
Ventricular Muscle ◽  
Cardiac Ventricular Muscle ◽  
Normal Human ◽  
Quadriceps Femoris Muscles

The intracellular distribution of acetylcholinesterase and butyrylcholinesterase in biopsied human rectus abdominis and quadriceps femoris muscles was investigated. Differential ultracentrifugal fractionation showed that the highest specific activity of acetylcholinesterase was associated with the microsomal fraction, this enzyme being concentrated 19-fold compared to the original homogenate. The highest specific activity of butyrylcholinesterase was found in the non-particulate fraction. The mitochondrial fraction showed the lowest specific cholinesterase activity. Wide variation of cholinesterase activity was found in various autopsied muscles, the highest being oculomotor muscle and the lowest cardiac ventricular muscle. About 90% of the cholinesterase activity of skeletal muscle was due to acetylcholinesterase and 10% to butyrylcholinesterase.

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