RHODOPSIN KINASE - SUBSTRATE-SPECIFICITY AND FACTORS THAT INFLUENCE ACTIVITY
RHODOPSIN KINASE - SUBSTRATE-SPECIFICITY AND FACTORS THAT INFLUENCE ACTIVITY
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DOI：
10.1021/bi00407a010
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发表时间：
1988-04-05
期刊：
影响因子：
2.9
通讯作者：
HARGRAVE, PA
中科院分区：
文献类型：
--
作者：
PALCZEWSKI, K;MCDOWELL, JH;HARGRAVE, PA
用Sitaramayya [Sitaramayya，A.（1986）Biochemistry 25，5460]，进行了一些小的修改。该酶能够磷酸化盘膜中的牛视紫红质、来自其他物种的视紫红质和溶解在温和洗涤剂（十二烷基麦芽糖苷）中的视紫红质。视紫红质激酶可以磷酸化含有来自牛视紫红质的适当序列的合成肽;然而，这些肽的Km值比视紫红质或ATP高约3个数量级。视紫红质胞质表面的一些肽抑制磷酸化。这些结果表明，不止一个区域的视紫红质参与相互作用的激酶。Mg 2+是Mg-ATP复合物所必需的，如观察到的（乙二胺四乙酸）抑制激酶活性所示。第二，高于复合所有存在的ATP所需浓度的游离Mg 2+激活激酶。第三，较高浓度的Mg 2+产生Mg-ATP-Mg而不是Mg-ATP，因此抑制激酶活性。其他生理上重要的阳离子如Ca 2+、Na+和K+可能通过形成金属离子-ATP复合物，从而降低Mg-ATP的浓度，从而降低激酶的活性。5“-[对-（氟磺酰基）苯甲酰基]腺苷（FSO 2BzAdo）是一种激酶和ATP酶抑制剂，对视紫红质激酶的抑制作用符合准一级动力学。一阶常数与FSO_2BzAdo浓度呈双曲线关系。这表明ATP类似物和酶之间的可逆复合物在类似物共价连接至视紫红质激酶之前形成。当在饱和浓度下使用时，Mg-ATP和ATP几乎完全保护激酶免于失活。Mg 2+和视紫红质只有轻微的保护作用。这些结果表明，修饰发生在激酶活性位点。
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. Mg-ATP and ATP almost completely protect the kinase against inactivation when used at saturating concentrations. Mg2+ and rhodopsin have only a minor protective effect. These results suggest that modification occurs at the kinase active site.