Molnar E, Varga S, Martonosi A
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Biochim Biophys Acta. 1991 Sep 10;1068(1):17-26. doi: 10.1016/0005-2736(91)90056-e.
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.
在1 mM钒酸盐存在的情况下，用紫外光照射肌浆网囊泡会导致Ca(2+)-ATP酶光裂解成两个片段（Vegh等人，(1990) 《生物化学与生物物理学报》1023, 168 - 183）。在没有Ca2+的情况下，光裂解发生在分子靠近磷酸受体天冬氨酸-351的N端一半区域。在2 mM Ca2+存在的情况下，光裂解转移到ATP酶的C端一半区域，靠近异硫氰酸荧光素（FITC）结合位点（赖氨酸-515）。当在2 mM CaCl2存在下照射时，约一半的Ca(2+)-ATP酶迅速裂解，同时伴随着ATP酶活性几乎完全不可逆的丧失；该酶的进一步裂解缓慢，主要影响在Ca2+存在下产生的C端片段。用C12E8溶解Ca(2+)-ATP酶不会影响任何一种构象下的光裂解位点。钒酸盐诱导的Ca(2+)-ATP酶晶体在光裂解过程中被破坏，而针对磷酸化位点（PR - 8）和FITC结合区域（PR - 11）的抗ATP酶抗体的结合增强。在约一半的Ca(2+)-ATP酶被裂解的条件下，牛肾Na+,K(+)-ATP酶对光裂解不敏感。猪胃H+,K(+)-ATP酶在长时间照射后轻微裂解产生的片段与Ca(2+)-ATP酶的片段不同。
