N-myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase.

Recoverin, a recently identified member of the EF-hand superfamily of Ca(2+)-binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N-terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N-acyl group. To clarify the role of the N-terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non-myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 microM) for the myristoylated and non-myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non-myristoylated form (EC50 are 0.9 and 6.5 microM, respectively).

Calcium-sensitive control of rhodopsin phosphorylation in the reconstituted system consisting of photoreceptor membranes, rhodopsin kinase and recoverin.

Rhodopsin phosphorylation in the reconstituted system consisting of urea-washed photoreceptor membranes, rhodopsin kinase and recoverin is regulated by Ca2+: the process takes place at low [Ca2+] but is suppressed at high [Ca2+]. In the absence of recoverin, rhodopsin kinase is active irrespective of the cation concentration used. Hence, recoverin is an inhibitor (at high [Ca2+]) but not an activator of rhodopsin kinase. Based jointly on these data obtained on the reconstituted system and on our preceding experiments on rod outer segments suspension, one may conclude that (i) the function of recoverin in retina rod cells is the Ca(2+)-sensitive control of rhodopsin phosphorylation and (ii) the presence of recoverin is essential and sufficient to provide rhodopsin kinase with the Ca2+ sensitivity.

Recoverin mediates the calcium effect upon rhodopsin phosphorylation and cGMP hydrolysis in bovine retina rod cells.

Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod outer segments are Ca2+ dependent in the presence of ATP. The level of rhodopsin phosphorylation decreases and the lifetime of active phosphodiesterase increases when the free [Ca2+] is raised from < 1 nM to about 1 microM; in both cases the half-maximal effect was observed at 140-170 nM of free Ca2+. Antibodies to recoverin reverse both effects at high [Ca2+] but have no influence at low [Ca2+]. We conclude that the Ca2+ effects observed are mediated by recoverin which inhibits rhodopsin kinase at a high Ca2+ level.

[Function of the calcium-binding protein p26 (recoverin) in bovine retinal rods]. / Izuchenie funktsii kal'tsiisviazyvaiushchego belka p26 (rekoverina) v palochkakh setchatki byka.

The bovine retina rod cell protein with an apparent M(r) 26 kDa (p26 [1] or recoverin [2]) was suggested to be a calcium-sensitive regulator of photoreceptor guanylate cyclase. The data obtained show that highly purified p26 is not capable of restoring guanylate cyclase in washed ROS membranes up to the level of a ROS suspension. At the same time we found that a Ca(2+)-sensitive complex of p26 and a protein with apparent M(r) 67 kDa, presumably rhodopsin kinase, is present in the ROS extract. We suggest that rhodopsin kinase can be a functional target for p26 in retina rod cells.

The presence of a calcium-sensitive p26-containing complex in bovine retina rod cells.

A protein with apparent M(r) 26 kDa (p26 [(1991) Biokimya 56, 225-228] or recoverin [(1991) Science 251, 915-918]) was suggested to activate GC in a calcium dependent manner in bovine retina rod cells [(1991) Science 251, 915-918; (1991) EMBO J, 10, 793-798]. However, according to our present data homogeneous p26 preparations do not activate the enzyme. At the same time we have revealed a complex of p26 with an unidentified protein, presumably RK, in bovine ROS. Calcium favours formation of the complex whereas EGTA addition (which corresponds to a low free Ca2+ concentration) leads to its dissociation.

[Effect of radioprotectors on lipid peroxidation in liver microsomes induced by ultraviolet irradiation of the skin in rats]. / Vliianie radioprotektorov na perekislenie lipidov mikrosom pecheni, indutsirovannoe ul'trafioletovym oblucheniem kozhi krys.

The modifying effect of radioprotectors (serotonin, cysteamine, ionol) on lipid peroxidation intensification of liver microsomes caused by rat skin ultraviolet radiation has been studied. A possible mechanism of action of these compounds on the investigated indexes during their preventive injection is under discussion.

[Effect of ultraviolet rays on peroxidation and its regulation in the rat liver]. / Vliianie ul'trafioletovogo izlucheniia na protsess perekisnogo okisleniia i ego reguliatsiiu v pecheni krys.

The indirect effect of rat skin ultraviolet (UV) irradiation on lipid peroxidation and enzymatic systems of the liver has been studied. The processes of lipid peroxidation have been intensified after 72 hours of UV-irradiation, which is evidently due both to the activation of enzymatic system of initiation and propagation of lipid peroxidation and to the parallel decrease of the activity of enzymatic system regulation of given process in liver.

[Effect of ionizing radiation on O2 generation by cytochrome P-450]. / Vliianie ioniziruiushchei radiatsii na generatsiiu O2 tsitokhromom P-450.

A study was made of generation of superoxide anion-radical (O2-) by cytochrome P-450 in a microsomal membrane of rat liver. Using spectrophotometry (by oxidation of adrenaline to adrenochrome) and ESR (with a spin-trap, tiron) the authors showed the ability of O2- generation by P-450 through decomposition of organic peroxides. During the first 24 h following irradiation of rats with doses of 7 and 10 Gy, the generation of O2- by cytochrome P-450 of rat liver microsomes was increased. Mechanisms of the postirradiation modification of O2- generation rate are discussed.

[Effect of x-irradiation on the generation of O2- in rat liver microsomes]. / Vliianie rentgenovskogo oblucheniia na generatsiiu O2- v mikrosomakh pecheni krys.

A study was made of generation of O2- in NADPH-dependent chain of oxidation of liver microsomes in irradiated rats (7 and 10 Gy). The rate of O2- generation sharply increased at early times after irradiation. The data obtained prompt an assumption that the increase in the rate of O2- generation is perhaps connected with the changes in functioning of both NADPH-cytochrome P-450-reductase and cytochrome P-450.