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1.
Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar. 相似文献
2.
A T Tsin 《The Journal of experimental zoology》1986,238(3):311-317
The kinetics of esterification of exogenous retinol by cell membranes prepared from the crude homogenate of the frog retinal pigment epithelium was studied. The formation of retinyl palmitate from added retinol was directly assayed by high performance liquid chromatography (HPLC). A linear relationship was observed between the amount of protein (up to 2 mg) in the incubation medium and the amount of retinyl palmitate formed. At room temperature, this reaction took less than 2 hours to complete. By varying the substrate concentration in the incubation medium, the reciprocal of initial velocity of the reaction (nmol retinyl palmitate formed per hour) was plotted against the reciprocal of substrate concentration (nmol of retinol). This double-reciprocal plot shows that the apparent Km of the reaction was 10 microM with an apparent Vmax of 9.1 nmol of retinyl palmitate per hour per mg protein. When this assay was repeated in the presence of 3,4-didehydroretinol (20 microM), the kinetics of the reaction showed the pattern of that of a competitive inhibitor, suggesting that 3,4-didehydroretinol competes with retinol for the same active site for esterification. The esterification of 3,4-didehydroretinol resulted in the formation of 3,4-didehydroretinyl palmitate, which was also measured by HPLC. The amount of 3,4-didehydroretinyl palmitate formed by this reaction decreased in proportion to increased retinol concentration in the incubation mixture. This further confirms that a competition exists between the esterification of retinol and 3,4-didehydroretinol by retinal pigment epithelium of the frog. 相似文献
3.
When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells. 相似文献
4.
A.Catharine Ross 《Analytical biochemistry》1981,115(2):324-330
Individual long-chain fatty acid esters of retinol can be resolved by high-performance liquid chromatography using an octyl- or phenyl-substituted reverse-phase column and mixtures of acetonitrile with water as mobile phase. This simple procedure provides good resolution of biologically important retinyl esters including retinyl palmitate and retinyl oleate. Using an isocratic elution system, it is shown that nine synthetic esters of retinol, ranging in fatty acyl chain length from 12 to 20 carbons, each elute with a unique elution volume and produce an absorbance signal at 340 nm proportional to molar concentration. The method is suitable for analysis of various esters of retinol in biological samples including lymph chylomicrons and blood plasma. The octyl-substituted reverse-phase column can also be used to separate more polar neutral retinoids including retinol and retinaldehyde. 相似文献
5.
S Takase T Goda 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,96(2):415-419
1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation. 相似文献
6.
S Hartmann O Froescheis F Ringenbach R Wyss F Bucheli S Bischof J Bausch U.-W Wiegand 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,751(2):939
A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data. 相似文献
7.
F J Zieve 《Analytical biochemistry》1973,51(2):436-442
A procedure has been developed for the separation of labeled fatty acids from tri-, di-, and monoglycerides using small disposable columns of TEAE-cellulose. This procedure is used as the basis of a lipase assay which is rapid, sensitive and unaffected by wide variations in the composition of the reaction mixtures. 0.5 nmole [14C]oleic acid can be detected, and the entire procedure requires less than 3 min. 相似文献
8.
Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine 总被引:4,自引:0,他引:4
Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme. 相似文献
9.
Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo 总被引:3,自引:0,他引:3
A clear separation of retinol, retinal, and retinoic acid has been achieved by liquid-gel partition chromatography on Sephadex LH-20 with solvent mixtures of chloroform, Skellysolve B, and methanol. A mixture of retinyl esters, retinol, retinal, and retinoic acid has been resolved on hydroxyalkoxypropyl Sephadex using Skellysolve B and acetone. There is no decomposition of any of the vitamin A compounds during chromatography, and recovery is complete. The combination of mildness and potential for resolution makes liquid-gel partition chromatography a superior tool for the separation of vitamin A compounds. This method has been applied to the study of vitamin A metabolism at physiological levels in the vitamin A-deficient rat. Retinyl palmitate, an ester of retinoic acid, retinal, retinol, retinoic acid, and a polar metabolite have been demonstrated in various tissues of the rat 12 hr after a dose of 2 micro g of [11-(14)C]retinyl acetate. 相似文献
10.
A study was conducted to investigate the effects of a simulated weightlessness induced by chronic immobilization on vitamin A status. To simulate the stress condition of weightlessness, rats were suspended for 10 days in a special jacket to which metal chains were attached. Animals received a commercial stock diet. Control rats were pair-fed in reference to the suspended rats. As compared with the control, prolonged immobilization resulted in a decrease in body weight gain and an increase in adrenal weight occurred. In the suspended rats, serum concentrations of retinol and retinol-binding protein (RBP) declined. Hepatic retinyl palmitate content increased, and the hepatic retinol level was decreased. The prolonged immobilization led to significantly reduced retinyl palmitate levels in the testis and lung as well as lowered testicular retinol levels. The results suggest that the stress state induced by prolonged immobilization caused accumulation of hepatic retinyl palmitate, decreasing the serum retinol concentration and retinyl ester content in the extrahepatic tissues. 相似文献
11.
A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction. 相似文献
12.
Vitamin A metabolism in the human intestinal Caco-2 cell line 总被引:2,自引:0,他引:2
The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein 总被引:1,自引:0,他引:1
We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls. 相似文献
14.
The visual cells, visual pigments and major retinoids of the Mongolian jird (Meriones unguiculatus) were examined. Light and electron microscope analyses show that these jirds had mainly rod photoreceptors. Octylglucoside extracts prepared from their retinas contained only rhodopsin with a maximum absorption at 497 nm and a concentration of 0.51 nmol per retina. Employing a standard method of high performance liquid chromatography (HPLC), the pigment epithelium from each eye was found to possess 0.52 nmol of retinyl palmitate (the most abundant form of retinyl ester) along with a small amount of retinol (0.02 nmol). Most of the retinoids in the body of these animals are stored in the liver, in the form of retinyl palmitate (1228.80 nmol per gram liver). As the Mongolian jird is small, inexpensive and readily available, this animal is a mammalian species suitable for the research of the biochemistry of retinoids and vision. 相似文献
15.
Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder. 相似文献
16.
A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 microM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and stearate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59 +/- 3%, S.E.) and kidney (76 +/- 3%), with considerably lower overall activity in kidney (57-375 nmol/h per g of tissue) than in liver (394-1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 micrograms/dl, and of liver vitamin A concentrations from 0 to 32 micrograms/g. Pig liver retinyl ester hydrolase differs from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability. 相似文献
17.
Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction. 相似文献
18.
M D Ball H C Furr J A Olson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1986,84(4):513-517
Acyl coenzyme A:retinol acyltransferase activity was identified in the microsomes from a polar bear liver. The highest rate of in vitro retinol esterification was 821 pmol/min/mg microsomal protein. The in vitro esterification rate displayed a small dependence upon the concentration of exogenous protein (bovine serum albumin) and even less on the concentration of sulfhydryl-reducing agent (dithiothreitol). Vitamin A was present in the liver at a concentration of 8050 micrograms/g tissue, with 98% of the vitamin in its ester form. Retinyl palmitate was 37.3% of the total liver retinyl esters, while retinyl oleate represented 20.9%, stearate 12.8%, and linoleate 7.7%. 相似文献
19.
Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.
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The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function. 相似文献
20.
John C. Saari Sidney Futterman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,444(3):789-793
Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder. 相似文献