Solubilization, purification, and reconstitution of the sodium-calcium exchanger from bovine retinal rod outer segments

We have previously described a method for the solubilization and reconstitution of the cGMP-gated cation channel from the membranes of bovine rod outer segments (Cook, N. J., Zeilinger, C., Koch, K.-W., and Kaupp, U. B. (1986) J. Biol. Chem. 261, 17033-17039). Here we report that not only cGMP but also sodium is capable of releasing entrapped calcium from liposomes reconstituted with total rod outer segment membrane proteins. Other alkali cations tested were unable to induce calcium efflux; therefore, we concluded that the sodium-induced calcium efflux was due to the sodium-calcium exchanger. Sodium was found to activate calcium efflux from these liposomes with an EC50 of approximately equal to 35 mM, comparable to values reported for the sodium-calcium exchanger in native rod outer segment membranes. We found that reconstitution of the sodium-calcium exchanger is quantitative and used this method to assay the exchange protein during purification using conventional protein chromatographic techniques. In this way, we were able to purify and identify as the rod outer segment sodium-calcium exchanger a glycoprotein of apparent Mr = 220,000 to greater than 90% homogeneity. The specific activity of the purified protein at room temperature was 8.2 mumol of Ca2+ exchanged min-1 mg-1 of protein at 50 mM Na+, corresponding to a turnover number of approximately equal to 30 Ca2+ (or 90 Na+) s-1 exchanger-1. The Mr = 220,000 protein reported here appears to be distinct from another protein ("rim protein") with an identical Mr known to exist in these membranes.     

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Phosphoinositide synthesis in bovine rod outer segments

Phosphoinositide turnover has been implicated in signal transduction in a variety of cells, including photoreceptors. We demonstrate here the presence of a complete pathway for rapid synthesis of phosphoinositides in isolated bovine retinal rod outer segments (ROS) free of microsomal contaminants. Synthesis was measured by the incorporation of label from radioactive precursors, [gamma-32P]ATP and [3H]inositol. [gamma-32P]ATP also produced large amounts of labeled phosphatidic acid. Incorporation of [3H]inositol required CTP and Mn2+. Mn2+ increased 32P incorporation into phosphatidylinositol 4-phosphate, while spermine increased phosphoinositide labeling generally. ROS that had been washed to remove soluble and peripheral proteins incorporated less label than unwashed ROS into phosphatidic acid and phosphatidylinositol. No effects of light were detected. Inhibitory effects of high concentrations of nonhydrolyzable GTP analogues were probably due to competition with ATP.     

Purification of retinol dehydrogenase from bovine retinal rod outer segments

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.     

Characterization of rhodopsin kinase purified from bovine rod outer segments

Rhodopsin kinase was purified by sequential chromatography on DEAE-cellulose and blue-Sepharose. Kinase activity co-purified with a 62-kDa polypeptide, which bound light-dependently in the absence of ATP to purified vesicle-reconstituted rhodopsin. Purified rhodopsin kinase is free of any detectable arrestin or the retinal G-protein. Rhodopsin kinase is autophosphorylated on serine residues which is unaffected by the presence of bleached rhodopsin and results in a transition in molecular mass to 64 kDa. Autophosphorylation of the kinase did not appear to alter the overall rate of rhodopsin phosphorylation or the apparent KM (0.6 microM) for purified reconstituted rhodopsin. Peptides corresponding to sequences within opsin loops 3-4 and 5-6 and the COOH terminus inhibited kinase phosphorylation of bleached rhodopsin, suggesting at least three potential sites to account for the stable high affinity binding of rhodopsin kinase to the bleached photoreceptor molecule that are at least in part distinct from the substrate sites for phosphorylation. These sequences are similar to those proposed for receptor recognition of G-proteins and indicate that the domains involved in light-dependent binding of rhodopsin kinase and retinal G-protein are similar or overlapping.     

The membrane bound retinol dehydrogenase from bovine rod outer segments

Retinol dehydrogenase from bovine rod outer segments was solubilized in detergent and partially purified 25-fold through a combination of hydroxyapatite and retinyl-Sepharose chromatography. Alltrans retinol solubilized in protein solutions of bovine serum albumin or β-lactalbumin was a better substrate for the enzyme than retinol solubilized in detergents or suspended in buffer. Retinol dehydrogenase was sensitive to the carbonyl reagent pyridoxal-5′-phosphate but was not inhibited by retinal followed by reduction with NaBH₄. The solubilized enzyme requires phospholipids to maintain enzymatic activity, as was evidenced by the inactivating effect of phospholipase A₂ on the partially purified enzyme.     

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Calcium-hydrogen exchange in isolated bovine rod outer segments

We have measured Ca-H exchange in rod photoreceptors with different preparations of rod outer segments isolated from bovine retinas (ROS). One preparation contained ROS with an intact plasma membrane (intact ROS), and in the other preparation, the plasma membrane was leaky to small solutes (leaky ROS) and the cytoplasmic space was freely accessible to externally applied solutes. Addition of Ca2+ to Ca2+-depleted ROS (both intact and leaky) resulted in uptake of Ca2+ that was accompanied by the release of protons when catalytic amounts of the ionophore A23187 were present. This ionophore mediates Ca-H exchange transport across ROS membranes and serves to gain access to the intracellular compartment where Ca-H exchange appears to take place. Two protons were ejected for each calcium ion taken up. Conversely, when protons were added to Ca2+-enriched ROS, Ca2+ was released in the presence of A23187. The majority of this Ca-H exchange was observed only when A23187 was present in both intact and leaky ROS. We conclude that Ca-H exchange occurs predominantly in the intradiskal space and at the surface of the disk membrane rather than across the disk membrane. These exchange binding sites can accommodate 10 mol of Ca2+/mol of rhodopsin at physiological pH. We were unable to detect any Ca2+ release when a proton gradient was rapidly established across the disk membrane in the absence of A23187. These results are discussed in relation to the hypothesis that protons produced by the light-induced hydrolysis of cGMP cause the release of Ca2+ into the cytoplasm of rod photoreceptor cells.     

Protein kinase activity of bovine retina rod outer segments

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.     

Superoxide dismutase of bovine and frog rod outer segments.

Bovine rod outer segments (ROS) contain soluble superoxide dismutase (SOD) which from cyanide sensitivity and electrophoretic mobility appears identical to CuZn SOD of erythrocytes. Enzyme activity of ROS extracts is 200–400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activate lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.     

Light sensitive zinc content of protein fractions from bovine rod outer segments

Bovine retinas, isolated rod outer segments and emulphogene extracts of rod outer segments have been shown to contain appreciable amounts of Zn²⁺, Ca²⁺ and Mg²⁺ when isolated in the absence of added metal ions. Chromatography of emulphogene extracted rod outer segments in Sephadex G-25 showed virtually all the Ca²⁺, Zn²⁺ and protein to elute with the void volume. Levels of Zn²⁺ but not Ca²⁺ were light sensitive. The Zn²⁺ contents of protein fractions were about 60% higher when samples were bleached. Under optimal conditions protein fractions contained 1.4 – 1.8 g atoms Zn²⁺/mole rhodopsin for dark adapted samples and 2.1 to 3.2 g atoms Zn²⁺/mole of rhodopsin for bleached samples.     

Phosphorylation of endogenous proteins of bovine retina rod outer segments

The components of bovine rod outer segments (ROS) and water-soluble extracts of ROS were separated by SDS-electrophoresis after incubation with [gamma-32P]ATP or [gamma-32P]GTP at different experimental conditions. After that gels were autoradiographed to reveal the phosphorylated intermediates. Our results suggest, that ROS contains the following protein kinase systems: 1) water-soluble cAMP-dependent protein kinases, that uses ATP, but not GTP, and phosphorylates the water-soluble 30 000 molecular weight protein; 2) protein kinase that uses GTP (probably, ATP also) and phosphorylates the 20 000 molecular weight protein in light-adapted ROS; 3) water-soluble cyclic nucleotide- and Ca2+-independent protein kinase that uses ATP rather than GTP and phosphorylates the water-soluble 70 000 molecular weight protein. The concentrations of phosphorylated intermediates in bovine ROS are estimated.     

Adenylate kinase activity in rod outer segments of bovine retina

The rod outer segments of bovine retina contain two different adenylate kinases: a soluble activity, which is not sensitive to calcium ion, and an activity bound to disk membranes, which is dependent on the calcium levels. In fact, the maximal activity associated to the disks is reached at Ca(2+) concentrations between 10(-6) and 10(-7) M, which is the range of calcium level actually present in the rod cell. The Michaelis-Menten kinetics of the enzyme activity on disk membranes was determined and the actual concentrations of ATP, AMP and ADP were measured in the photoreceptor outer segment. Therefore, the physiological relevance of the adenylate kinase activity was discussed considering the above results. The formation of ATP catalyzed by the enzyme seems appropriate to supply at least some of the reactions necessary for phototransduction, indicating that ATP could be regenerated from ADP directly on the disk membranes where the photoreception events take place.     

Solubilization and functional reconstitution of a chloride channel fromTorpedo californica electroplax

Summary Chloride channels were detergent-extracted fromTorpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thawsonication procedure. Concentrative uptake of³⁶Cl^–, driven by a Cl^– gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl^– over cations, is blocked by SCN^–, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl^–>Br^–>F^–>SO ₄ ^2– , as does the voltagegated Cl^– channel fromTorpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity.     

Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments

A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogeneous after purification and exhibits a molecular mass of approximately 85 kDa, as determined by SDS/PAGE. From its chromatographic behaviour on hydroxyapatite, and from Western-blotting experiments using isoenzyme-specific antibodies, we were able to identify the bovine rod outer segment protein kinase C as being of the alpha or type-III form. The purified protein kinase C has a specific activity of 1066 nmol 32P.min-1.mg protein-1, and shows a 30-fold activation upon the addition of the effectors Ca2+, PtdSer and 1,2-diacylglycerol. Arachidonic acid and linoleic acid were also found to enhance significantly the activity of the purified enzyme.     

Calcium ion-regulated phospholipase C activity in bovine rod outer segments

Bovine retinal rod outer segment membranes are enriched in a phosphoinositide-specific phosphodiesterase (phospholipase C) activity strictly modulated by free calcium ion concentration. The enzyme(s) was highly active on phosphatidylinositol 4,5-bisphosphate: maximal hydrolysis rate was attained at 10(-5)M Ca2+ and accounted for 91 +/- 4 nmoles hydrolyzed/min/mg of protein. The results support the notion that in vivo the enzyme(s) is regulated so as to conform to the phototransduction events.