Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.
Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.
Tate
C A
CA
Department of Medicine, Baylor College of Medicine, Methodist Hospital, Houston, Texas.
Shin
G
G
Walseth
T F
TF
Taffet
G E
GE
Bick
R J
RJ
Entman
M L
ML
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D006868:Hydrolysis; D009206:Myocardium / Q000378:metabolism*; D010777:Photochemistry; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013379:Substrate Specificity; D013861:Thiocyanates / Q000378:metabolism*
D000345:Affinity Labels; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate