Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum.
The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.
Seidler
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Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
Jona
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I
Vegh
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Martonosi
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A
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology; D005753:Gastric Mucosa / Q000201:enzymology; D007211:Indoles / Q000494:pharmacology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009183:Mycotoxins / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D007211:Indoles; D009183:Mycotoxins; D010446:Peptide Fragments; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; C000543:cyclopiazonic acid