Acylation of disc membrane rhodopsin may be nonenzymatic

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.     

Strong Association of Unesterified [3H]Docosahexaenoic Acid and [3H-Docosahexaenoyl]Phosphatidate to Rhodopsin During In Vivo Labeling of Frog Retinal Rod Outer Segments

Docosahexaenoic acid (DHA, 22:6n-3), the most prevalent fatty acid in phospholipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [³H]DHA-lipids to rhodopsin in ROS from frogs (Rana pipiens) after in vitro (4 hrs) and in vivo (1 day and 32 days) labeling. Lipids from lyophilized ROS were sequentially extracted with hexane (neutral lipids), chloroform:methanol (phospholipids) and acidified chloroform:methanol (acidic phospholipids). After in vitro labeling, free [³H]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rhodopsin). In contrast, after in vivo labeling free [³H]DHA was mainly recovered in the acidic solvent extract (89–99%). Of all phospholipids, [³H-DHA]phosphatidic acid (PA) displayed the highest binding to rhodopsin after both in vitro (43% in acidic extract) and in vivo (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

RENEWAL OF FATTY ACIDS IN THE MEMBRANES OF VISUAL CELL OUTER SEGMENTS

The renewal of fatty acids in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with tritiated palmitic, stearic, or arachidonic acids. Most of the radioactive material could be extracted from the retina with chloroform-methanol, indicating that the fatty acids had been esterified in lipids. Analysis of the extracts, after injection of [³H]palmitic acid, revealed that the radioactivity was predominantly in phospholipid. Palmitic acid was initially concentrated in the pigment epithelium, particularly in oil droplets which are storage sites for vitamin A esterified with fatty acid. The cytoplasm, but not the nucleus of these cells, was also heavily labeled. Radioactive fatty acid was bound immediately to the visual cell outer segment membranes, including detached rod membranes which had been phagocytized by the pigment epithelium. This is believed to be due to fatty acid exchange in phospholipid molecules already situated in the membranes. Gradually, the concentration of radioactive material in the visual cell outer segment membranes increased, apparently as a result of the addition of new phospholipid molecules, possibly augmented by the transfer from the pigment epithelium of esterified vitamin A. Injected fatty acid became particularly concentrated in new membranes which are continually assembled at the base of rod outer segments. This localized concentration was short-lived, apparently due to the rapid renewal of fatty acid. The results support the conclusion that rods renew the lipids of their outer segments by membrane replacement, whereas both rods and cones renew the membrane lipids by molecular replacement, including fatty acid exchange and replacement of phospholipid molecules in existing membranes.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

Cell-free acylation of rat brain myelin proteolipid protein and DM-20.

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.     

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Recycling of docosahexaenoic acid in rat retinas during n-3 fatty acid deficiency.

About 50% of the fatty acids in retinal rod outer segments is docosahexaenoic acid [22:6(n-3)], a member of the linolenic acid [18:3(n-3)] family of essential fatty acids. Dietary deprivation of n-3 fatty acids leads to only modest changes in 22:6(n-3) levels in the retina. We investigated the mechanism(s) by which the retina conserves 22:6(n-3) during n-3 fatty acid deficiency. Weanling rats were fed diets containing 10% (wt/wt) hydrogenated coconut oil (no n-3 or n-6 fatty acids), linseed oil (high n-3, low n-6), or safflower oil (high n-6, less than 0.1% n-3) for 15 weeks. The turnover of phospholipid molecular species and the turnover and recycling of 22:6(n-3) in phospholipids of the rod outer segment membranes were examined after the intravitreal injection of [2-3H]glycerol and [4,5-3H]22:6(n-3), respectively. Animals were killed on selected days, and rod outer segment membranes, liver, and plasma were taken for lipid analyses. The half-lives (days) of individual phospholipid molecular species and total phospholipid 22:6(n-3) were calculated from the slopes of the regression lines of log specific activity versus time. There were no differences in the turnover rates of phospholipid molecular species among the three dietary groups, as determined by the disappearance of labeled glycerol. Thus, 22:6(n-3) is not conserved through a reduction in phospholipid turnover in rod outer segments. However, the half-life of [4,5-3H]22:6(n-3) in the linseed oil group (19 days) was significantly less than in the coconut oil (54 days) and safflower oil (not measurable) groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active labeling of phosphatidylcholines by [1-14C]docosahexaenoate in isolated photoreceptor membranes

Isolated bovine rod outer segments and photoreceptor disks actively incorporated [1-14C]docosahexaenoate (22:6) into phospholipids when incubated in the presence of CoA, ATP, and Mg2+. About 80% of the esterified fatty acid was in phosphatidylcholine (PC). Microsomal and mitochondrial fractions incorporated as much 22:6 as rod outer segments, but it was distributed among various phospholipids and neutral glycerides. The isolated photoreceptor membrane thus contains an acyl-CoA synthetase which activates the fatty acid and a docosahexaenoyl-CoA-lysophosphatidylcholine acyltransferase activity. The specific radioactivity of PC was higher in rod outer segments than in the other subcellular fractions. About 2/3 of the label in photoreceptor membrane PC was in its dipolyunsaturated molecular species and 1/3 in hexaenes. Dipolyunsaturated PCs showed high turnover rates of 22:6 in all three subcellular membranes, especially in mitochondria. Retinal membranes in vitro seem to take up free [14C]22:6 from the medium by simple diffusion or partition into the membrane lipid. The ability of these membranes to activate and esterify [1-14C]22:6 indicates that docosahexaenoate-containing molecular species of retina lipids, including those of photoreceptor membranes, are subject to acylation-deacylation reactions in situ.     

In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Incorporation of glucosamine into rhodopsin in isolated bovine retina

Radioactive glucosamine is incorporated into the outer segments of the rod cells of bovine retinas incubated in vitro. One component of the outer segment labeled in this process is rhodopsin which can be extracted with detergent, purified by sequential chromatography on calcium phosphate-Celite and agarose, and shown to be light sensitive by its altered chromatographic mobility. The radioactive component can be released from rhodopsin by acid hydrolysis and shown to migrate with glucosamine on paper chromatography. In double label experiments both glucosamine and leucine are incorporated into rhodopsin. The time course of glucosamine incorporation is similar to that of leucine. The system supports prolonged synthesis of both the polypeptide and oligosaccharide portions of the rhodopsin molecule in vitro.     

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.     

Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Phosphorylated proteins may play an important role in regulating the metabolism or function of rod photoreceptors. In mammalian retinas, a photoreceptor protein of 33 000 (33K) molecular weight is phosphorylated in a cyclic nucleotide dependent manner in vitro. Since light initiates the activation of a photoreceptor-specific phosphodiesterase and a rapid reduction in guanosine cyclic 3',5'-phosphate concentration, phosphorylation of the 33K protein may be modulated by light in situ. In order to test this possibility, dark-adapted rat retinas were incubated for 30 min in the dark in phosphate-free Kreb's buffer containing [32P]orthophosphate. Following incubation, rod outer segments were detached by shaking, and the 32P-labeled rod outer segment proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and quantitated by densitometric scanning. The incorporation of radioactivity (32P) into the 33K protein was higher than into any other rod outer segment protein, and the amount of 32P-labeled 33K protein in the detached rod outer segments remained unchanged during 10 additional min of darkness. The addition of isobutylmethylxanthine to the incubation medium enhanced the incorporation of 32P into 33K protein to about 400% of the original level. Exposure of freshly detached rod outer segments to room light for 90 s decreased the amount of labeled 33K protein to 45% of its original level. The dephosphorylation of labeled 33K protein continued, reaching 12% of the original dark value 10 min after the previously illuminated sample was returned to darkness. Light initiated the phosphorylation of rhodopsin, and rhodopsin phosphorylation continued during the postillumination period of darkness.     

Control of light-activated phosphorylation in frog photoreceptor membranes.

In this paper, we examine some factors which regulate the efficiency of light in activating rhodopsin phosphorylation. We have measured phosphate incorporation after illumination in suspensions of bullfrog rod outer segments incubated with [gamma-32P]ATP. We observed that delaying ATP addition after illumination causes maximum phosphate incorporation to decrease 80% within 2 h. This decay occurs in urea-treated, extracted rod outer segment membranes. The decay of the light effect is not influenced by regeneration of opsin to rhodopsin or the presence of long-lived photoproducts. However, regeneration of opsin increases the amount of phosphorylation initiated by a second exposure to light. Further phosphorylation can also occur after phosphate groups have been removed from the membranes by dephosphorylation. Finally, we have confirmed our earlier observation that small amounts of light (bleaching less than 5% of the rhodopsin present) are more effective, by tenfold, in initiating phosphorylation than are larger amounts.     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Myristyl and palmityl acylation of the insulin receptor

The presence of covalently bound fatty acids in the insulin receptor has been explored in cultured human (IM-9) lymphocytes. Both alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor incorporate [3H]myristic and [3H]palmitic acids in a covalent form. The effects of alkali and hydroxylamine on the labeled subunits indicate the existence of two different kinds of fatty acid linkage to the protein with chemical stabilities compatible with amide and ester bonds. The alpha subunit contains only amide-linked fatty acid while the beta subunit has both amide- and ester-linked fatty acids. Analysis by high performance liquid chromatography after acid hydrolysis of the [3H]myristate- and [3H]palmitate-labeled subunits demonstrates the fatty acid nature of the label. Furthermore, both [3H]myristic and [3H]palmitic acids are found attached to the receptor subunits regardless of which fatty acid was used for labeling. The incorporation of fatty acids into the insulin receptor is dependent on protein synthesis and is also detectable in the Mr = 190,000 proreceptor form. Fatty acylation is a newly identified post-translational modification of the insulin receptor which may have an important role in its interaction with the membrane and/or its biological function.