Differences in the susceptibility of various cation transport ATPases to vanadate-catalyzed photocleavage.
Illumination of sarcoplasmic reticulum vesicles by ultraviolet light in the presence of 1 mM vanadate causes photocleavage of the Ca(2+)-ATPase into two fragments (Vegh et al. (1990) Biochim. Biophys. Acta 1023, 168-183). In the absence of Ca2+ the photocleavage occurs in the N-terminal half of the molecule near the phosphate acceptor Asp-351. In the presence of 2 mM Ca2+ the photocleavage shifts to the C-terminal half of the ATPase, near the FITC binding site (Lys-515). About half of the Ca(2+)-ATPase was cleaved rapidly, accompanied by nearly complete, irreversible loss of ATPase activity when illuminated in the presence of 2 mM CaCl2; further cleavage of the enzyme was slow and affected primarily the C-terminal fragment produced in the presence of Ca2+. Solubilization of the Ca(2+)-ATPase with C12E8 did not affect the site of photocleavage in either conformation. The vanadate-induced Ca(2+)-ATPase crystals were disrupted during photocleavage, while the binding of anti-ATPase antibodies directed against the phosphorylation site (PR-8) and against the FITC binding region (PR-11) was enhanced. The bovine kidney Na+,K(+)-ATPase was insensitive to photocleavage under conditions where about half the Ca(2+)-ATPase was fragmented. The slight cleavage of the pig gastric H+,K(+)-ATPase after prolonged illumination produced fragments that are distinct from the fragments of the Ca(2+)-ATPase.
Molnar
E
E
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Varga
S
S
Martonosi
A
A
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000276:immunology; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004797:Enzyme-Linked Immunosorbent Assay; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D017506:H(+)-K(+)-Exchanging ATPase; D008969:Molecular Sequence Data; D010777:Photochemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D013861:Thiocyanates; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology*
D000906:Antibodies; D005452:Fluoresceins; D013861:Thiocyanates; D014638:Vanadates; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate