No abstract

Intracellular Ca2+ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca2+ pump and that a Ca(2+)-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca2+ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca2+ homeostasis and pump activity.

Trans-plasma membrane electron transport is critical for maintaining cellular redox balance and viability, yet few, if any, investigations have studied it in intact primary neurons. In this investigation, extracellular reduction of 2,6-dichloroindophenol (DCIP) and ferricyanide (FeCN) were measured as indicators of trans-plasma membrane electron transport by chick forebrain neurons. Neurons readily reduced DCIP, but not FeCN unless CoQ 1 , an exogenous ubiquinone analog, was added to the assays. CoQ 1 stimulated FeCN reduction in a dosedependent manner but had no effect on DCIP reduction. Reduction of both substrates was totally inhibited by e-maleimidocaproic acid (MCA), a membrane-impermeant thiol reagent, and slightly inhibited by superoxide dismutase. Diphenylene iodonium, a flavoenzyme inhibitor, completely inhibited FeCN reduction but had no affect on DCIP reduction, suggesting that these substrates are reduced by distinct redox pathways. The relationship between plasma membrane electron transport and neuronal viability was tested using the inhibitors MCA and capsaicin. MCA caused a dose-dependent decline in neuronal viability that closely paralleled its inhibition of both reductase activities. Similarly capsaicin, a NADH oxidase inhibitor, induced a rapid decline in neuronal viability. These results suggest that trans-plasma membrane electron transport helps maintain a stable redox environment required for neuronal viability.

No abstract

Pig gastric microsomal vesicles enriched in gastric H+,K+-ATPase and K+-pNPPase were digested with bee venom phospholipase A2 at 21 or 37 degrees C. The unattacked phospholipids were then related to the remaining enzyme activities, followed by reconstitution with microsomal phospholipids and the endogenous activator protein. Gastric K+-stimulated ATPase was nearly abolished within 10 min of phospholipase A2 treatment. A substantial amount of pNPPase activity remained unaffected under identical conditions. About 80% of the microsomal phosphatidylethanolamine was attacked by phospholipase A2 at both temperatures while 60 and 79% of the phosphatidylcholine was hydrolyzed at 21 and 37 degrees C, respectively. Analysis of the phospholipids revealed that phospholipase A2 attacked only the phosphatidylcholine and phosphatidylethanolamine molecules enriched in polyunsaturated fatty acids. Microsomal H+,K+-ATPase system inactivated by phospholipase A2 at 21 degrees C could be largely restored by the endogenous activator alone. On the other hand, those inactivated at 37 degrees C needed pretreatment with phosphatidylcholine before assaying with the activator protein for maximal reconstitution; phosphatidylethanolamine was totally ineffective in restoration of the enzyme activity. Analysis of the fatty acid composition of the lysophosphatidylcholine following phospholipase A2 treatment at 21 and 37 degrees C suggested involvement of some phosphatidylcholine molecules relatively enriched in saturated fatty acids and extremely poor in polyunsaturated fatty acids in gastric ATPase function. The data not only pointed out the importance of phosphatidylcholine and the endogenous activator in gastric microsomal H+,K+-ATPase reaction but also demonstrated considerable heterogeneity within the same species of microsomal phospholipids.