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PubMed1995

Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

Acylation↗
PubMed1995

Amino-terminal myristoylation induces cooperative calcium binding to recoverin.

Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca(2+)-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 microM. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 microM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca(2+)-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca2+ binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 microM, as in unmyristoylated recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and < 0.05 for unmyristoylated recoverin. Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully liganded protein.

Allosteric Site↗
PubMed2006

Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase.

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Animals↗
PubMed2006

Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin.

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).

Amino Acid Sequence↗
PubMed1993

Role of the acylated amino terminus of recoverin in Ca(2+)-dependent membrane interaction.

Recoverin, a calcium ion (Ca2+)-binding protein of vertebrate photoreceptors, binds to photoreceptor membranes when the Ca2+ concentration is greater than 1 micromolar. This interaction requires a fatty acyl residue covalently linked to the recoverin amino (NH2)-terminus. Removal of the acyl residue, either by proteolytic cleavage of the NH2-terminus or by production of nonacylated recoverin, prevented recoverin from binding to membranes. The acylated recoverin NH2-terminus could be cleaved by trypsin only when Ca2+ was bound to recoverin. These results suggest that the hydrophobic NH2-terminus is constrained in Ca(2+)-free recoverin and liberated by Ca2+ binding. The hydrophobic acyl moiety of recoverin may interact with the membrane only when recoverin binds Ca2+.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome.

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR(+) cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24(+) CAR(+) cancer patients. In addition, the CTL responses were obtained from three of ten CAR(-) cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR(+) cancer patients and that of CAR(-) cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR(+) cancer patients. Moreover, in cases other than CAR(+) cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24(+) cancer patients with a recoverin-expressing tumor.

Antigens, Neoplasm↗
PubMed1998

Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy (CAR) syndrome. In these studies we examined the effect of anti-recoverin antibodies from the sera of patients with CAR and rat monoclonal antibody on the retinas of Lewis rats. Anti-recoverin autoanti-bodies penetrated into the photoreceptor and bipolar cell layers following intravitreal injection. Their presence in the retina could be detected by immunofluorescence 24 h after injection. At the same time, individual cells undergoing apoptosis were identified throughout photoreceptor and bipolar cell layers using terminal transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopy. Normal antibodies used in control experiments did not produce TUNEL labeling. At 24 h, DNA fragmentation was confirmed by DNA ladder electrophoresis. At the electron microscopic level, there was clear evidence of cells undergoing apoptotic cell death in the retinas treated with anti-recoverin antibodies. At 24 and 96 h, nuclear chromatin condensation and increased vacuolization of photoreceptor outer segments were observed. An examination of retinas from animals receiving anti-retinal antibodies revealed a loss of 1-2 rows of nuclei in the outer and inner nuclear layers whereas all controls (sham, normal IgG, phosphate buffered saline) showed an unchanged number of nuclei rows. In addition, there was an increase in spacing between the rows of nuclei of the outer nuclear layer in retinas treated with anti- recoverin antibodies, indicating additional cell loss. These studies provide clear evidence that anti-recoverin antibodies are capable of penetrating photoreceptor and bipolar cells, the normal site of recoverin expression in the retina, and that anti-recoverin antibodies produce apoptotic cell death. A similar mechanism may occur in patients with CAR, which may lead to visual loss and blindness.

Animals↗
PubMed2005

Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level.

Recoverin is cotranslationally modified by the covalent linkage of a myristoyl group to its N terminus. It is a member of a family of Ca(2+)-myristoyl switch proteins. Recombinant myristoylated revoverin is currently produced by the cotransformation of bacteria with recoverin and an enzyme that allows N-myristoylation and by supplementing the culture medium with myristic acid. A large variation in the myristoylation level of recoverin and in the amount of myristic acid supplied to the culture medium can be found in the literature. Moreover, although it is known to strongly affect bacterial growth, the amount of ethanol used to solubilize myristic acid is only scarcely mentioned. To improve our understanding of the parameters responsible for recombinant recoverin myristoylation, the effects of myristic acid and ethanol on recoverin myristoylation and expression levels have been systematically studied. In addition, a single-step purification procedure to produce purified myristoylated and nonmyristoylated recombinant recoverin has also been devised. Finally, sodium myristate has been used as an efficient alternative substrate to achieve high myristoylation and expression levels of recoverin. Given that a large number of proteins are myristoylated, these procedures could be applied to several other proteins in addition to recoverin.

Animals↗
PubMed2004

Stimulation of the aberrant expression of a paraneoplastic antigen, recoverin, in small cell lung cancer cell lines.

Recoverin, a retina-specific Ca2+-binding protein, is one of the paraneoplastic antigens (PNAs) which are normally present in neurons, but can also be aberrantly expressed in malignant tumors localized outside the nervous system. In this study, we have analyzed 16 small cell lung carcinoma (SCLC) and 12 non-small cell lung carcinoma cell lines and found that none of them is capable of expressing recoverin in vitro. However, two small cell lung carcinoma lines, NCI-H69 and NCI-H82, became recoverin-positive after cultivation in the presence of butyrate. Recoverin expression in the butyrate-treated cells has been detected by immunoblotting with polyclonal (monospecific) antibodies against recoverin and confirmed by the analysis of recoverin mRNA expression. To our knowledge, this work is the first to demonstrate stimulation of the aberrant expression of recoverin in cancer cell lines in vitro. This result opens the way to investigation of the mechanisms underlying the aberrant expression of recoverin, as well as other paraneoplastic antigens, in tumor cells.

Antigens, Neoplasm↗
PubMed1997

Calcium-dependent binding of recoverin to membranes monitored by surface plasmon resonance spectroscopy in real time.

Recoverin is an N-myristoylated Ca2+-binding protein that serves as a Ca2+-sensor in visual transduction. We studied the dynamics of its Ca2+-dependent membrane association which depends on the myristoyl modification (Ca2+-myristoyl switch) by surface plasmon resonance spectroscopy. Either recoverin or phospholipid vesicles were immobilized on a sensor chip surface, and the respective binding partner was supplied in the mobile phase. Binding of recoverin to artificial liposomes or rod outer segment membranes was strictly dependent on Ca2+ and the myristoyl group. The Ca2+-myristoyl switch was half-maximal between 4.0 and 7.7 microM Ca2+, depending on whether recoverin or liposomes were in the mobile phase. At saturating [Ca2+], the dissociation constant (KD) of recoverin for phospholipid liposomes was approximately 150 microM. The association and dissociation of recoverin to membranes was fast and biphasic (fast and slow components) with time constants on the order of 0.1 s-1 and 0.01 s-1, respectively. Dissociation of the recoverin-membrane complex was 3-fold faster at low than at high free [Ca2+]. We discuss the analogy between the liposome-sensor chip and the disk surface and conclude that a minor fraction of the total recoverin in a rod outer segment is associated with membranes at resting dark levels of free [Ca2+].

Animals↗
PubMedDate not supplied

Recoverin is a zinc-binding protein.

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.

Anilino Naphthalenesulfonates↗
PubMed1996

Expression of a photoreceptor protein, recoverin, as a cancer-associated retinopathy autoantigen in human lung cancer cell lines.

Recently, a photoreceptor protein, recoverin, has been recognised as an autoantigen of cancer-associated retinopathy (CAR), a rare paraneoplastic neurological syndrome often associated with patients with small-cell lung cancer (SCLC). Although until quite recently the specific expression of recoverin in cancer cells had not been indicated, Polans et al. (Polans AS, Witkowska D, Haley TL, Amundson D, Baizer L, Adamus G 1995, Proc. Natl. Acad. Sci. USA, 92, 9176-9180) demonstrated the specific expression of recoverin in lung tumour and primary cultured tumour cells from a CAR patient. We examined the expression of recoverin in human lung cancer cell lines by reverse transcription polymerase chain reaction (PCR), Northern blotting and Western immunoblotting. Recoverin was expressed in only one SCLC cell line from a patient with CAR. The sequence of recoverin cDNA from the cells was identical to the human recoverin sequence. These findings strongly support the hypothesis that the ectopic expression of wild-type recoverin in SCLC induces the cancer-retina immunological cross-reaction, leading to visual loss in CAR.

Biomarkers, Tumor↗
PubMed1992

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Amino Acid Sequence↗
PubMed1993

Recoverin alters its surface properties depending on both calcium-binding and N-terminal myristoylation.

The solution structure and calcium-dependent structural changes of recoverin, a 23 kDa calcium binding protein of vertebrate photoreceptors, have been studied by small-angle X-ray scattering and CD, as well as the effect of N-terminal myristoylation. The CD spectrum is not affected by N-terminal myristoylation, but strongly affected by Ca2+, indicating that N-terminal myristoylation alone does not cause a conformational change. The major conformational change in recoverin induced by Ca2+ is characterized as a decrease in the alpha-helical content of the protein and an increase in global size upon removal of Ca2+. In the presence of Ca2+, unmyristoylated recoverin is monomeric and globular in solution, while N-terminal myristoylation brings about aggregation. In the absence of Ca2+, unmyristoylated recoverin tends to aggregate, while myristoylated recoverin becomes monomeric and globular. These observations indicate that recoverin changes its surface properties depending on both calcium binding and N-terminal myristoylation. Melittin interacts non-specifically only with the myristoylated recoverin in the absence of Ca2+. This may be indicative of the properties of the interaction between recoverin and its normal physiological target enzyme.

Calcium↗
PubMed1994

Genetic and physical mapping of human recoverin: a gene expressed in retinal photoreceptors.

PURPOSE: Recoverin is a calcium-binding protein that may be involved in phototransduction in mammalian retinal photoreceptors, and is considered to be a candidate gene for retinitis pigmentosa. This study was undertaken to develop the recoverin locus into a polymorphic marker for future linkage studies on retinitis pigmentosa families. METHODS: A human genomic cosmid clone was isolated and used to map the recoverin gene to a human chromosome through hybridization to a panel of somatic hybrid cell line DNAs, and to human metaphase chromosomes by fluorescence in situ hybridization. A dinucleotide repeat polymorphism located within the coding region of the recoverin gene was identified, and used to genetically map the recoverin gene relative to index markers. In addition, three restriction fragment length polymorphisms revealed by the cosmid clone were identified and characterized. RESULTS: Hybridization to the somatic hybrid cell line DNAs localized the recoverin gene to chromosome 17. Recoverin was further localized to 17p12-p13 by fluorescence in situ hybridization. The dinucleotide repeat polymorphism and restriction fragment length polymorphisms at the recoverin locus have a cumulative polymorphic information content = 0.71. CONCLUSIONS: These polymorphic markers and additional closely linked markers will be useful for linkage analysis of families with retinitis pigmentosa.

Base Sequence↗
PubMed1994

Inhibition of rhodopsin phosphorylation by non-myristoylated recombinant recoverin.

Bovine recoverin regulates rhodopsin phosphorylation and controls photoreceptor light sensitivity in a Ca(2+)-dependent manner. Recoverin is post-translationally modified with lipids (myristic acid or related lipids) at its N-terminus. Since with this lipid modification (N-myristoylation), recoverin associates with rod outer segment membranes in a Ca(2+)-dependent manner, N-myristoylation has been suggested to be important for the function of this protein. To study the role of this modification, we obtained recombinant non-myristoylated recoverin in E. coli and studied its functional properties. Here, we report that recombinant non-myristoylated recoverin inhibits rhodopsin phosphorylation at Ca2+ concentrations of 30 nM-10 microM in a similar way as native N-myristoylated recoverin does. Thus, our result showed that N-myristoylation is not essential for the Ca(2+)-dependent inhibition of rhodopsin phosphorylation by recoverin.

Adenosine Triphosphate↗
PubMed1997

Early expression of recoverin in a unique population of neurons in the human retina.

The calcium-binding protein recoverin has been reported as present in photoreceptors, cone bipolar cells and sparse cells in the ganglion cell layer in the adult retinae of various vertebrate species. The present study was undertaken to clarify the developmental pattern of recoverin-immunoreactive cells in the human retina with particular attention to the cells in the inner retinal layers. In the adult human retina, small populations of recoverin-containing cells are present in the ganglion cell and nerve fiber layers. However, the precursors of these cells are quite numerous on the inner and outer borders of the nerve fiber layer in the fetal retina. By 13 weeks of gestation these cells express recoverin very intensely. By 24 weeks they are mature-looking with relatively large soma sizes (mean = 118 microns 2) and appear round, oval or multipolar in shape, with varying numbers of short processes. There follows a noticeable reduction of the mean soma size, but little change in morphology and process number during the remaining gestational stages up to and after birth. The mean numerical density of the recoverin-positive cells in the fetal inner retinal layers is gradually reduced from the high level at 13 weeks until birth, when there is a great drop to the adult level. The recoverin-immunoreactive cells in the ganglion cell layer demonstrate distinctively different developmental and morphological features from the principle neurons and glial cells in the retina. They are probably the neurons derived from the marginal zone of the retinal primordium that reside in the inner and outer borders of the nerve fiber layer due to the invasion of ganglion cell axons. The expression of recoverin in the neurons may be significant in maintaining an inside-out and centroperipheral gradient of calcium concentration in the premature retina, thereby playing a role in determining the polarity of the differentiating ganglion cells and the growth of their axons in a centrifugal spatiotemporal order.

Antibody Specificity↗
PubMed1995

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).

Animals↗