Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes.
Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.
Hicks
D B
DB
Yocum
C F
CF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology; D000458:Cyanobacteria / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D007425:Intracellular Membranes / Q000201:enzymology; D008027:Light; D000432:Methanol / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D012997:Solvents; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D000432:Methanol