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R R Rando

Publications and source records attributed to R R Rando.

At least 91 records · Page 5Linked to original sources
PubMed1990

The gamma subunit of transducin is farnesylated.

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification.

Animals↗
PubMed1989

Schiff-base deprotonation is mandatory for light-dependent rhodopsin phosphorylation.

The absorption of light by rhodopsin leads to the formation of an activated intermediate (R*) capable of catalysing the exchange of GTP for GDP in a retinal guanine-nucleotide-binding regulatory protein (transducin). The ability of R* to function as a catalyst is terminated by the rhodopsin kinase. The 10 nonactive-site lysine residues of rhodopsin can be reductively dimethylated to form permethylated rhodopsin (PMRh). This derivative is phosphorylated to the same extent as rhodopsin after photolysis. The monomethylation of the active-site lysine residue of PMRh yields active-site-methylated rhodopsin (AMRh). It had previously been shown, by using AMRh, that the formation of R* and its spectroscopic signature metarhodopsin II requires the photochemically induced deprotonation of the active-site Schiff base [Longstaff, Calhoon & Rando (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4209-4213]. Here it is demonstrated that active-site Schiff-base deprotonation is also mandatory in the formation of the form of photolyzed rhodopsin that is susceptible to phosphorylation by rhodopsin kinase. In terms of the spectroscopically defined rhodopsin intermediates, this means that only metarhodopsin II and possibly metarhodopsin III are the actual substrates for rhodopsin kinase.

Animals↗
PubMed1989

Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation.

The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.

Animals↗
PubMed1989

The molecular basis of retinoic acid induced night blindness.

Drugs which affect the processing of vitamin A in the retina or pigment epithelium can cause ocular toxicity. It is shown here that the retinoic acids, which are used in the treatment of skin disorders and which cause night blindness, inhibit the ocular retinol dehydrogenases in an in vitro system. This is shown to lead to a decrease in the formation of the visual chromophore 11-cis-retinal, thus explaining why night blindness might occur.

Alcohol Oxidoreductases↗
PubMed1989

Solubilization and partial purification of retinyl ester synthetase and retinoid isomerase from bovine ocular pigment epithelium.

Studies reported previously from this laboratory have demonstrated that membranes from the pigment epithelium of the vertebrate eye can transform free all-trans-retinol to 11-cis-retinol as well as 11-cis- and all trans-retinyl esters (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853; Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) J. Biol. Chem. 262, 16848-16857; Fulton, B. S., and Rando, R. R. (1987) Biochemistry 26, 7938-7945). The congeneric retinals are also formed under conditions where retinol redox activity is present. Here we report the successful solubilization of both the retinyl ester synthetase and isomerase activities from the pigment epithelium membranes of the bovine eye. The zwitterionic detergent Zwittergent 3-14(N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; cmc 0.012%) gave optimal solubilization of both activities. Three initial criteria for successful solubilization were used. First, high speed centrifugation (greater than 150,000 x g) left the activities in the supernatant. Second, the solubilized enzymatic activities were found in the included volume upon gel filtration. Finally, the solubilized activities were quantitatively passed through a 0.22-microns filter. Employing anion exchange and gel filtration chromatography results in a partial purification of the retinyl ester synthetase (approximately 189-fold). The solubilized retinoid isomerase is also partially purified (approximately 10-14-fold) following anion exchange chromatography. It is also shown that the membrane-bound and solubilized ester synthetase catalyzes the esterification of retinol using added lecithins as exogenous acyl donors. In addition, evidence is provided indicating that there is a positional selectivity for the acyl group transfer from the lecithin to retinol. The transfer occurs largely, if not entirely, from the 1-position of the lecithin.

Acyltransferases↗
PubMed1989

Membranes as the energy source in the endergonic transformation of vitamin A to 11-cis-retinol.

The eye needs to biosynthesize 11-cis-retinoids because the chromophore of rhodopsin is 11-cis-retinal. The critical metabolic step is the endergonic isomerization of free all-trans-retinol (vitamin A) into 11-cis-retinol. This isomerization process can take place in isolated membranes from the retinal pigment epithelium in the absence of added energy sources. Specific binding proteins probably do not serve as an energy source, and since all of the reactions in the visual cycle are shown here to be reversible, trapping reactions also do not participate in the isomerization reaction. One previously unexplored possibility is that the chemical energy in the bonds of the membrane itself may drive the isomerization reaction. A group transfer reaction is proposed that forms a retinyl ester from a lipid acyl donor and vitamin A. This transfer can drive the isomerization reaction because the all-trans-retinyl ester is isomerized directly to 11-cis-retinol. Thus, the free energy of hydrolysis of the ester is coupled to the thermodynamically uphill trans to cis isomerization. The prediction of an obligate C-O bond cleavage in the vitamin A moiety during isomerization is borne out. Although the natural substrate for isomerization is not known, all-trans-retinyl palmitate is processed in vitro to 11-cis-retinol by pigment epithelial membranes.

Amphibians↗
PubMed1989

The active-site environment of rhodopsin.

The 11-cis-retinal binding site of rhodopsin is of great interest because it is buried in the membrane but yet must provide an environment for charged amino acids. In addition, the active-site lysine residue must be able to engage in rapid Schiff base formation with 11-cis-retinal at neutral and lower pH values. This requires that this lysine be unprotonated. We have begun to study the environment of the active-site lysine using a reporter group adducted to it. Non-active-site permethylated opsin was reacted with 5-nitrosalicylaldehyde, and the resulting Schiff base was permanently fixed by borohydride reduction. The stoichiometry of incorporation was one. This chromophoric and pH-sensitive reporter group affords information on the active-site environment of rhodopsin by determining the ionization constants of its ionizable groups at different pH values. The pH titration of the modified protein showed a single pKa = 7.8 +/- 0.19 ascribable to the ionization of the phenol. The ionization of the modified lysine residue was not observed at all pH values studied. These studies are interpreted to mean that a negatively charged amino acid is propinquous to the active-site lysine residue and that this latter residue does not have an unusually low pKa.

Aldehydes↗
PubMed1989

The interaction of cycloserine with pyruvate and other biologically relevant alpha-ketoacids.

The ability of cycloserine solutions to deplete alpha-oxoacids has been found to be correlated with the spontaneous transformation of cycloserine into a derivative dimer (2,5-bis-(aminoxymethyl)-3,6-diketopiperazine). Synthetic dimer was found to react rapidly with pyruvate to form the expected oxime. Two lines of evidence indicate that it is the cycloserine dimer and not cycloserine itself that reacts with alpha-ketoacid. First, the 1H NMR spectrum of the purified oxime is superimposable with that arising when the dimer and pyruvate are mixed and the spectrum taken immediately thereafter. Second, the mass spectrum of the reaction product of cycloserine dimer and methylpyruvate is totally consistent with the formation of a stable oxime derivative. Furthermore, when cycloserine is incubated with pyruvate the oxime derived from the dimer is found. These observations clearly indicate that cycloserine in solution can have chemical activities in addition to its ability to interfere with pyridoxal dependent reactions. On these grounds it is concluded that any biological action of cycloserine should be interpreted cautiously.

Carbon Dioxide↗
PubMed1989

Structural basis of protein kinase C activation by tumor promoters.

Protein kinase C (PKC) is an important enzyme that helps govern cell metabolism and growth. The enzyme is physiologically activated when an (S)-diglyceride binds to its own regulatory domain. The saturable binding site of the regulatory domain can also be bound by any of a group of structurally diverse tumor promoters, including debromoaplysiatoxins (DATs), phorbol esters, ingenols, teleocidins, and bryostatins. The question of how the same binding site can be the target of these structurally diverse molecules is of considerable importance and is addressed in this article. The relatively rigid structure of DAT and the fact that it possesses a diglyceride moiety renders it an ideal starting template. Structure-activity studies with PKC reveal that the C29 but not the C30 stereocenter of DAT is critical for activity. Furthermore, 3-deoxy-DAT and DAT are equipotent as PKC activators, hence the C3 hydroxyl group of DAT is not critical for activity. Straightforward structural considerations show that the C30 hydroxyl group of DAT matches the C3 hydroxyl group of diglyceride, the C29 stereocenter of DAT matches the C2 stereocenter of (S)-diglyceride, and the C1 ester moiety of DAT matches the C2 ester moiety of diglyceride. Based on these studies and on published structure-activity observations on other tumor promoters, a structural hypothesis is developed to account for the chemical mechanism of tumor promoter action. Experimentally testable predictions are made concerning the interactions with PKC of several classes of tumor PKC activators.

Animals↗
PubMed1988

Structural studies on the diglyceride-mediated activation of protein kinase C.

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.

Calcium↗
PubMed1988

Stereochemical inversion at C-15 accompanies the enzymatic isomerization of all-trans- to 11-cis-retinoids.

all-trans-Retinol (vitamin A) is processed by membranes from the pigment epithelium of the amphibian or bovine eye to form 11-cis-retinoids. When the isomerization reaction is performed with either [15(S)-3H,14C]-all-trans-retinol or [15(R)-3H,14C]-all-trans-retinol as substrate, the resultant 11-cis-retinals, formed by the in vitro enzymatic oxidation of the retinols, retain their 3H in the former case and lose it in the latter. The ocular all-trans- (pro-R specific) and 11-cis-retinol (pro-S specific) dehydrogenases operate with different stereochemistries with respect to the prochiral methylene hydroxyl centers of their substrates. Inversion of stereochemistry at the prochiral retinol centers was shown to accompany the isomerization process in both the amphibian and bovine systems. The 11-cis-retinol formed from [15(S)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(R)-3H,14C]-all-trans-retinol. The 11-cis-retinol formed from [15(R)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(S)-3H,14C]-all-trans-retinol. The stereochemistry at the prochiral center of retinol is not affected by the I2-catalyzed double-bond isomerization process and, hence, inversion of stereochemistry at C-15 must accompany isomerization. The same inverted stereochemistry was found with the associated retinyl palmitates. Possible mechanistic reasons for the observed inversion of stereochemistry during isomerization are discussed.

Alcohol Dehydrogenase↗
PubMed1988

Photochemistry of monomethylated and permethylated bacteriorhodopsin.

Methylation of the nonactive site lysines of bacteriorhodopsin to form permethylated bacteriorhodopsin does not interfere with the formation of the short wavelength intermediate M412 or light-induced proton release/uptake. The absorption spectrum is similar to that of the native bacteriorhodopsin. However, additional monomethylation of the active site lysine of bacteriorhodopsin causes a red shift of the absorption maximum from 568 nm in light-adapted bacteriorhodopsin [BR] to 630 nm. The photochemistry of active-site methylated BR does not proceed beyond the L-photointermediate. In particular, the photointermediate corresponding to M412 does not form, and there is no proton pumping. Moreover, there is no tyrosine deprotonation. Thus, the formation of an M-type photointermediate is required for proton pumping by BR.

Bacteriorhodopsins↗
PubMed1988

Regulation of protein kinase C activity by lipids.

Protein kinase C is activated by the simultaneous presence of phospholipid, a diglyceride, and Ca2+. Under physiological conditions the activity of the enzyme is regulated by the availability of diglycerides, which are the products of phosphoinositide hydrolysis. The phospholipid-kinase interactions appear not to be of a highly specific nature. Phosphatidylserine (PS) is presumed to be the endogenous lipid that interacts with the kinase, but other acidic lipids can substitute. On the other hand, the kinase-diglyceride interactions are highly specific in nature, as would be expected of a physiological regulator. These interactions are stereo-specific and stoichiometric with respect to diglyceride. The specificity is directed toward the glycerol backbone and hydrophilic oxygen moieties of the diglyceride. The removal of one or more of the oxygen atoms or the addition of a single methyl group to the glycerol backbone virtually abolishes the activity of a putative diglyceride activator. The extreme specificity of the kinase toward the diglycerides, however, must be contrasted with the abilities of structurally diverse tumor promotors and irritants to activate the kinase. Specific small-molecule antagonists of protein kinase C have yet to be developed. The small-molecule antagonists that have been developed so far have been relatively nonspecific cationic lipids that appear to function by interfering with the interaction between the acidic phospholipids and Ca2+.

Binding Sites↗
PubMed1987

Biochemical characterization of the retinoid isomerase system of the eye.

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.

Animals↗
PubMed1987

Biosynthesis of 11-cis-retinoids and retinyl esters by bovine pigment epithelium membranes.

Previously, we have shown that retina/pigment epithelium membranes from the amphibian can synthesize 11-cis-retinoids from added all-trans-retinol [Bernstein, P.S., Law, W.C., & Rando, R.R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853]. The activity was largely localized to the pigment epithelium. Here it is shown that, in the bovine system, the activity resides exclusively in the membranes of the pigment epithelium. Subcellular fractionation does not reveal a particular organelle where the activity resides. Washed bovine pigment epithelium membranes, which are devoid of retinoid redox activity, convert added all-trans-retinol to a mixture of 11-cis-retinol and its palmitate ester. all-trans-Retinal and all-trans-retinyl palmitate are not converted into 11-cis-retinoids by the membranes. The membranes show substantial ester synthetase activity, producing large amounts of all-trans-retinyl palmitate. Diverse chemical reagents, such as ethanol, hydroxylamine, and p-(hydroxymercuri)benzoate, inhibit both ester synthetase and isomerase activities in a roughly parallel fashion, suggesting a possible functional linkage between the two activities.

Animals↗
PubMed1987

Deprotonation of the Schiff base of bacteriorhodopsin is obligate in light-induced proton pumping.

Bacteriorhodopsin (bR) in purple membranes was permethylated with formaldehyde and pyridine-borane with the incorporation of approximately 12 methyl groups. This new pigment, PMbR, absorbed light in the dark-adapted state with a lambda max at 558 nm, virtually the same as that of bR. Light adaptation of PMbR produced a lambda max of 564 nm with a slightly elevated epsilon. Similar changes occurred with bR. When incorporated into asolectin vesicles, PMbR was able to pump protons in the light with an efficiency similar to that of bR itself. Bleaching of PMbR exposed its active site lysine residue, which was monomethylated to form active site methylated bR (AMbR) after regeneration with all-trans-retinal. This blue pigment, which is a cyanopsin rather than a rhodopsin, showed an extraordinary red shift, absorbing light with a lambda max of 620 nm in the dark-adapted state. Light adaptation of AMbR resulted in a spectral shift to 616 nm with a decrease in epsilon. This change was completely reversible in the dark. This shift was interpreted to mean that an L-like intermediate was accumulating, as would be expected if deprotonation of the protonated Schiff base could not occur to produce the M intermediate. Furthermore, when incorporated into asolectin vesicles, AMbR proved incapable of pumping protons in the light. It was concluded from these experiments that deprotonation of the Schiff base of bR is obligate for light-induced proton pumping.

Bacteriorhodopsins↗
PubMed1987

Mechanism of isomerization of 11-cis-retinal in lipid dispersions by aromatic amines.

It has previously been shown that retinotoxic, primary aromatic amines catalyze the isomerization of 11-cis-retinal to its all-trans congener after Schiff base formation [Bernstein, P.S., Fulton, B.S., & Rando, R.R. (1986) Biochemistry 25, 3370-3377]. This process led to the short-circuiting of the visual cycle and the observed retinotoxicity when it occurred in vivo. The catalysis was also observed to occur in vitro in phosphatidylcholine-based vesicles but not in hydrocarbon solutions. The rate of isomerization of an aromatic amine Schiff base of 11-cis-retinal in the phospholipid vesicles was typically 10(3)-fold more rapid than in hydrocarbon solutions. In this article, the mechanistic basis of this apparently membrane-specific catalysis is described. It was found that the rate enhancement effect observed was independent of the lipid used. Moreover, a bilayer structure was not important because rate enhancements were also observed in micelles. The rapid isomerization rates observed in lipid dispersions appear not be free radical initiated because free radical quenching agents, such as alpha-tocopherol and beta-carotene, had little effect on the isomerization rates. It was further found that aliphatic amines, such as n-dodecylamine, could be substituted for the aromatic amines in phospholipid. Finally, and most importantly, it was found that the isomerization of the aromatic amine retinal Schiff bases in phospholipid vesicles was acid-catalyzed. It is concluded that the rate enhancements observed for the isomerization of 11-cis-retinal-aromatic amine Schiff bases in lipid dispersions over that in hydrocarbon solvents are due to the occurrence of acid-base catalysis in the former.

Amines↗