Vanadate-catalyzed, conformationally specific photocleavage of the Ca2(+)-ATPase of sarcoplasmic reticulum.
Vanadate-sensitized photocleavage of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was observed upon illumination of sarcoplasmic reticulum vesicles or the purified Ca2(+)-ATPase by ultraviolet light in the presence of 1 mM monovanadate or decavanadate. The site of the photocleavage is influenced by the Ca2+ concentration of the medium. When the [Ca2+] is maintained below 10 nM by EGTA, the vanadate-catalyzed photocleavage yields fragments of approximately equal to 87 and approximately equal to 22 kDa, while in the presence of 2-20 mM Ca, polypeptides of 71 and 38 kDa are obtained as the principal cleavage products. These observations indicate that the site of the vanadate-catalyzed photocleavage is altered by changes in the conformation of Ca2(+)-ATPase. Selective tryptic proteolysis, at Arg-505-Ala-506, combined with covalent labeling of Lys-515 by fluorescein 5'-isothiocyanate and with the use of anti-ATPase antibodies of defined specificity, permitted the tentative allocation of the sites of photocleavage to the A fragment near the T2 cleavage site in the absence of Ca2+, and to the B fragment between Lys-515 and Asp-659 in the presence of 2-20 mM Ca2+. The loss of ATPase activity during illumination is accelerated by calcium in the presence of vanadate. The vanadate-catalyzed photocleavage in the presence of Ca2+ is consistent with the existence of an ATPase-Ca2(+)-vanadate complex (Markus et al. (1989) Biochemistry 28, 793-799).
Vegh
M
M
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Molnar
E
E
Martonosi
A
A
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000276:immunology / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010100:Oxygen; D010777:Photochemistry; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013861:Thiocyanates; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism / Q000494:pharmacology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D013861:Thiocyanates; D014638:Vanadates; D004533:Egtazic Acid; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D010100:Oxygen; D002118:Calcium