Palczewski K, McDowell J H, Hargrave P A
Department of Ophthalmology, University of Florida, Gainesville 32610-0284.
Biochemistry. 1988 Apr 5;27(7):2306-13. doi: 10.1021/bi00407a010.
Rhodopsin kinase was prepared from bovine retinas by the method of Sitaramayya [Sitaramayya, A. (1986) Biochemistry 25, 5460] with some minor modifications. The enzyme is able to phosphorylate bovine rhodopsin in the disk membrane, rhodopsin from other species, and rhodopsin solubilized in mild detergent (dodecyl maltoside). Rhodopsin kinase can phosphorylate synthetic peptides containing the appropriate sequences from bovine rhodopsin; however, the Km values for these peptides are about 3 orders of magnitude higher than that for rhodopsin or ATP. Some peptides from the cytosolic surface of rhodopsin inhibit the phosphorylation. These results suggest that more than one region of rhodopsin is involved in the interaction of rhodopsin of the kinase. Mg2+ is required for the Mg-ATP complex as shown by the observation that (ethylenedinitrilo)tetraacetic acid inhibits kinase activity. Second, free Mg2+ above the concentration required to complex all of the ATP present activates the kinase. Third, higher concentrations of Mg2+ yield Mg-ATP-Mg instead of Mg-ATP and therefore inhibit the kinase activity. Other physiologically important cations such as Ca2+, Na+, and K+ reduce the activity of the kinase, probably by forming a metal ion-ATP complex, thereby reducing the concentration of Mg-ATP. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo), an inhibitor of kinases and ATPases, inhibits rhodopsin kinase according to pseudo-first-order kinetics. The relationship between the first-order constant and the concentration of FSO2BzAdo is hyperbolic. This indicates that a reversible complex between the ATP analogue and the enzyme is formed prior to the covalent attachment of the analogue to rhodopsin kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
视紫红质激酶是通过西塔拉马亚（Sitaramayya, A. (1986) Biochemistry 25, 5460）的方法从牛视网膜中制备的，仅做了一些小的修改。该酶能够使盘膜中的牛视紫红质、其他物种的视紫红质以及溶解在温和去污剂（十二烷基麦芽糖苷）中的视紫红质磷酸化。视紫红质激酶可以使含有来自牛视紫红质适当序列的合成肽磷酸化；然而，这些肽的米氏常数（Km值）比视紫红质或ATP的米氏常数高约3个数量级。视紫红质胞质表面的一些肽会抑制磷酸化。这些结果表明，视紫红质的多个区域参与了与激酶的相互作用。如观察到（乙二胺四乙酸）抑制激酶活性所示，Mg2+是Mg-ATP复合物所必需的。其次，游离Mg2+高于使所有存在的ATP络合所需的浓度时会激活激酶。第三，更高浓度的Mg2+会产生Mg-ATP-Mg而不是Mg-ATP，因此会抑制激酶活性。其他生理上重要的阳离子，如Ca2+、Na+和K+，可能通过形成金属离子-ATP复合物来降低激酶的活性，从而降低Mg-ATP的浓度。激酶和ATP酶的抑制剂5'-[对-（氟磺酰基）苯甲酰基]腺苷（FSO2BzAdo）根据准一级动力学抑制视紫红质激酶。一级常数与FSO2BzAdo浓度之间的关系是双曲线的。这表明在该类似物与视紫红质激酶共价结合之前，ATP类似物与酶之间形成了可逆复合物。（摘要截短于250字）
