scholarly journals Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus

1970 ◽  
Vol 120 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Peter Owen ◽  
John H. Freer
Keyword(s):  
Succinate Dehydrogenase ◽  
Specific Activity ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Spectrophotometric Assay ◽  
Enzyme Activation ◽  
Succinate Dehydrogenase Activity ◽  
Micrococcus Lysodeikticus ◽  
Membrane Bound ◽  
Phenazine Methosulphate

1. Some properties of succinate dehydrogenase [succinate–(acceptor) oxidoreductase, EC 1.3.99.1] in membrane preparations from Micrococcus lysodeikticus (N.C.T.C. 2665) were investigated. 2. In the spectrophotometric assay system adopted the reaction velocity was shown to be proportional to the amount of membrane added. Dichlorophenol-indophenol, reduced photochemically in the presence of phenazine methosulphate, or enzymically by the membrane-bound enzyme, was shown to undergo reoxidation in the dark. 3. The membrane-bound enzyme was found to be inactivated at temperatures above 10°C. 4. The specific activity of membrane-bound succinate dehydrogenase was found to increase between two- and three-fold in diluted membrane preparations equilibrated at 0°C for 6h. Membranes treated with sodium deoxycholate showed no enzyme activation on dilution but displayed maximal activity, all activity being sedimentable at 103000g. The increase in specific activity observed on dilution could be partially inhibited by fixation with glutaraldehyde, or by the presence of bovine serum albumin. 5. The addition of Mg2+ or Ca2+ ions to membrane suspensions caused an overall depression of enzyme activity. 6. The results suggest the presence of an `inhibitor' that affects the expression of membrane bound succinate dehydrogenase activity.

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii

1975 ◽  
Vol 21 (11) ◽  
pp. 1807-1814 ◽  
Author(s):  
Peter Jurtshuk ◽  
John E. McEntire
Keyword(s):  
Electron Transport ◽  
Atpase Activity ◽  
Azotobacter Vinelandii ◽  
Specific Activity ◽  
Maximal Activity ◽  
Molar Ratio ◽  
Supernatant Fraction ◽  
Time Interval ◽  
Time Intervals ◽  
Membrane Bound

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 × g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activity occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0–60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment with trypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 °C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 °C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (−20 °C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.

ENZYMIC CHANGES IN THE CERVIX OF THE RAT AND HAMSTER DURING THE OESTROUS CYCLE AND THE EFFECT OF STEROIDS

1977 ◽  
Vol 72 (2) ◽  
pp. 153-161 ◽  
Author(s):  
ELIZABETH ZACHARIAH ◽  
N. R. MOUDGAL
Keyword(s):  
Alkaline Phosphatase ◽  
Specific Activity ◽  
Protein Biosynthesis ◽  
Hydrolytic Enzymes ◽  
Maximal Activity ◽  
Inhibitory Effect ◽  
Oestrous Cycle ◽  
Single Subcutaneous Injection ◽  
Arylsulphatase A ◽  
Phosphatase Alkaline

SUMMARY Changes in four hydrolytic enzymes, namely acid phosphatase, alkaline phosphatase, arylsulphatase A and B, of the cervix of the rat and hamster have been studied during the 4-day oestrous cycle. All four enzymes showed maximal activity on the day of oestrus and least activity on day 2 of dioestrus. All the enzymes showed significant reduction of activity after ovariectomy, arylsulphatase A and B showing the earliest changes in specific activity. A single subcutaneous injection of 0·02 μg oestradiol-17β/rat increased the specific activity of arylsulphatase A and B from the low ovariectomized level to that observed in control oestrous animals within 18 and 6 h respectively. A higher concentration of oestradiol-17β (2·0 μg) had an inhibitory effect. Progesterone was without effect on arylsulphatase B activity, but when given (2·0 mg) with 0·02 μg oestradiol-17β, it inhibited the response to oestrogen. Cycloheximide prevented the rise in arylsulphatase B activity occurring after oestrogen injection, suggesting a regulation of cervical arylsulphatase B at the level of protein biosynthesis. These results suggest that arylsulphatase B activity may be induced by oestrogen in the cervix of the rat.

Motoneuron and muscle fiber succinate dehydrogenase activity in control and overloaded plantaris

1991 ◽  
Vol 71 (4) ◽  
pp. 1589-1592 ◽  
Author(s):  
G. R. Chalmers ◽  
R. R. Roy ◽  
V. R. Edgerton
Keyword(s):  
Succinate Dehydrogenase ◽  
Muscle Fiber ◽  
Muscle Fibers ◽  
Cell Types ◽  
Mitochondrial Proteins ◽  
Succinate Dehydrogenase Activity ◽  
Fiber Size ◽  
The Right ◽  
Gastrocnemius Muscles ◽  
Selective Increase

To determine the level of coordination in succinate dehydrogenase (SDH) activity between plantaris motoneurons and muscle fibers, the soleus and gastrocnemius muscles were bilaterally excised in four cats to subject the plantaris to functional overload (FO). Five normal cats served as controls. Twelve weeks after surgery the right plantaris in each cat was injected with horseradish peroxidase to identify plantaris motoneurons. SDH activity then was measured in a population of plantaris motoneurons and muscle fibers in each cat. Control motoneurons and muscle fibers had similar mean SDH activities and a similar relationship between cell size and SDH activity. After FO, muscle fiber size doubled and mean muscle fiber SDH activity halved. Motoneuron mean SDH activity and size were unaffected by FO. Total SDH activity was unchanged in both the motoneurons and muscle fibers after FO. These changes suggest a selective increase in contractile proteins with little or no modulation of mitochondrial proteins in the muscle fibers, because total SDH activity was unchanged in muscle fibers after FO. These data demonstrate that although mean SDH activities were similar in control motoneurons and muscle fibers, mean SDH activities in these two cell types can change independently.

ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Phospholipase A2 ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Human Platelets ◽  
Phospholipid Bilayer ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Enzyme Substrate ◽  
Membrane Bound ◽  
Layer Chromatography

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

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