Characterization of Phenylmaleimide Inhibition of the Ca 2+ - ATPase from Skeletal-Muscle Sarcoplasmic Reticulum I. Velasco-Guille ´n, J. C. Go ´mez-Ferna ´ ndez, and J. A. Teruel 1 Departamento de Bioquı ´mica y Biologı ´a Molecular A, Facultad de Veterinaria, Campus de Espinardo, Universidad de Murcia, 30100 Murcia, Spain Received June 23, 1999, and in revised form August 23, 1999 The Ca 2 -ATPase from sarcoplasmic reticulum re- acts with phenylmaleimide, producing the inhibition of the ATPase activity following a pseudo-first-order kinetic with a rate constant of 19 M 1 s 1 . Calcium and ATP binding are not altered upon phenylmaleimide inhibition. However, the presence of millimolar cal- cium, and to a lesser extent magnesium, in the inhibi- tion medium enhances the effect of phenylmaleimide, causing a higher degree of inhibition. Solubilization with C 12 E 8 does not affect the ATPase inhibition, ex- cluding any kind of participation of the lipid bilayer. Phosphorylation with ATP in steady-state conditions as well as phosphorylation with inorganic phosphate in equilibrium conditions were strongly inhibited. Conversely, we have found that the occupancy of the phosphorylation site by ortovanadate fully protects against the inhibitory effect of phenylmaleimide, indi- cating a conformational transition associated with the phosphorylation reaction. © 1999 Academic Press Key Words: calcium ATPase; sarcoplasmic reticulum; maleimides; phenylmaleimide. The Ca 2+ -ATPase of skeletal muscle SR 2 is a P-type pump and utilizes the energy of the hydrolysis of one molecule of ATP to transport two calcium ions from the cytoplasmic side to the lumen of the SR (1, 2). This process is activated by calcium binding to the high- affinity calcium binding sites, and then phosphate transfer from bound ATP to Asp 351 causes formation of a phosphoenzyme intermediate, EP (3– 6). The EP can also be formed by inorganic phosphate in the absence of calcium by reversal of the catalytic cycle (7, 8). Vana- date can inhibit the Ca 2+ -ATPase by competing with phosphate, forming an EV intermediate analogous to that of phosphate (9 –13). Cysteine residues of the Ca 2+ -ATPase have been a target for different chemical reagents for probing the tertiary structure of the protein and possible conforma- tional changes as well as the implication of these sites in the function of the enzyme (14 –24). These reagents used toward cysteine residues are iodoacetamides, ma- leimides, and NBD-Cl. Iodoacetamides have been shown to specifically react with Cys 670 and Cys 674 without loss of transport activity (25). Since these Cysteines are very close they have been considered as the same site, which is located distant from the nucleotide binding site (25). The first maleimide described to react with the Ca 2+ - ATPase was NEM, which binds to Cys 344 and Cys 364 (26, 27), but under certain experimental conditions NEM can label Cys 377 and Cys 614 (28). Other maleim- ides have also been used, like fluorescent probes such as rhodamine maleimide, fluorescein maleimide, ANS maleimide, and DAB maleimide (23, 24). Fluo- rescein maleimide has been reported to bind mostly to Cys 471 and Cys 364 , and DAB maleimide to Cys 364 (29). (1,4-Phenylen)bismaleimide inhibits the Ca 2+ - ATPase interfering with the phosphorylation reac- tion and has been proposed to crosslink Cys 377 and Cys 614 . NBD-Cl specifically binds to Cys 344 and not to Cys 364 (30), allowing determination of intermolecular distances by fluorescence energy transfer to the nu- cleotide binding site (24). 1 To whom correspondence should be addressed. Fax: +3468364147. 2 Abbreviations used: AMP-PNP, adenyl-5'-imidodiphosphate; ANS maleimide, 2-(4'-maleimidoanilino)naphtalene-6-sulfonic acid; C 12 E 8 , dodecyl octaethylene glycol monoether; DAB maleimide, 4-dimethylamino-phenylazophenyl-4'-maleimide; EP, phosphoen- zyme; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-mor- pholino)propanesulfonic acid; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole; NEM, N-ethylmaleimide; SR, sarcoplasmic reticulum; Tea, triethanolamine. 0003-9861/99 $30.00 121 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 372, No. 1, December 1, pp. 121–127, 1999 Article ID abbi.1999.1464, available online at http://www.idealibrary.com on