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1.
Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.  相似文献   

2.
Liver alcohol dehydrogenase (E.C.1.1.1.1) is an NAD+/NADH dependent enzyme with a broad substrate specificity being active on an assortment of primary and secondary alcohols. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones as well as the oxidation of certain aldehydes to their related carboxylic acids. Although the bioinorganic and bioorganic aspects of the enzymatic mechanism, as well as the structures of various ternary complexes, have been extensively studied, the kinetic significance of certain intermediates has not been fully evaluated. Nevertheless, the availability of computer-assisted programs for kinetic simulation and molecular modeling make it possible to describe the biochemical mechanism more completely. Although the true physiological substrates of this zinc metalloenzyme are unknown, alcohol dehydrogenase effectively catalyzes not only the interconversion of all-trans-retinol and all-trans-retinal but also the oxidation of all-trans-retinal to the corresponding retinoic acid. Retinal and related vitamin A derivatives play fundamental roles in many physiological processes, most notably the vision process. Furthermore, retinoic acid is used in dermatology as well as in the prevention and treatment of different types of cancer. The enzyme-NAD+-retinol complex has an apparent pKa value of 7.2 and loses a proton rapidly. Proton inventory modeling suggests that the transition state for the hydride transfer step has a partial negative charge on the oxygen of retinoxide. Spectral evidence for an intermediate such as E-NAD+-retinoxide was obtained with enzyme that has cobalt(II) substituted for the active site zinc(II). Biophysical considerations of water in these biological processes coupled with the inverse solvent isotope effect lead to the conclusion that the zinc-bound alkoxide makes a strong hydrogen bond with the hydroxyl group of Ser48 and is thus activated for hydride transfer. Moderate pressure accelerates enzyme action indicative of a negative volume of activation. The data with retinol is discussed in terms of enzyme stability, mechanism, adaptation to extreme conditions, as well as water affinities of substrates and inhibitors. Our data concern all-trans, 9-cis, 11-cis, and 13-cis retinols as well as the corresponding retinals. In all cases the enzyme utilizes an approximately ordered mechanism for retinol–retinal interconversion and for retinal–retinoic acid transformation.  相似文献   

3.
A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min?1 mg?1, a K m of 1.8 μM, and a k cat of 1.7 min?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD + as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l?1, and 2,200 mg all-trans-retinal l?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l?1 h?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.  相似文献   

4.
A new prenyltransferase which catalyzes the synthesis of geranyl pyrophosphate as the only product from dimethylallyl pyrophosphate and isopentenyl pyrophosphate has been separated from other known prenyltransferases from Micrococcuslysodeikticus. This enzyme fraction is also capable of synthesizing all-trans geranylgeranyl pyrophosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate though it lacks ability to synthesize farnesyl pyrophosphate.  相似文献   

5.
The key step in the visual pigment regeneration process is an enzyme-catalyzedtrans tocis retinoid isomerization reaction. This reaction is of substantial general interest, because it requires the input of metabolic energy. The energy is needed because the 11-cis-retinoid reaction products are approximately 4kcal/mol higher in energy than their all-trans congeners. In the retinal pigment epithelium a novel enzymatic system has been discovered which is capable of converting all-trans-retinol into all-trans retinyl esters, by means of a lecithin retinol acyl transferase (LRAT), followed by the direct processing of the ester into 11-cis-retinol. In this process the free energy of hydrolysis of a retinyl ester, estimated to be approximately –5kcal/mol, is coupled to the endothermic (+4kcal/mol) isomerization reaction, resulting in an overall exothermic process. The overall process is analogous to ATP-dependent group transfer reactions, but here the energy is provided by the membrane phospholipids. This process illustrates a new role for membranes: they can serve as an energy source.  相似文献   

6.
Pyrene lecithin, a new excimer-forming lipid molecule, has been synthesized to examine the transversal mobility of probe molecules in lecithin bilayer vesicles. The rate of the lipid exchange is obtained by following the excimer yield as a function of time after mixing of fluorescence doped and undoped vesicles. A rapid exchange (τ12 = 11 s) is followed by a slow transfer (t12 = 8 h). Above the lipid phase transition the fast transfer can be attributed to an exchange of lipid molecules from the outer layer of one vesicle to the outer layer of another one. The slow exchange is interpreted in terms of the ‘flip-flop’ process between the two layers of a single bilayer vesicle.Using pyrene and pyrene decanoic acid as probe molecules only the fast transfer through the water phase is observed (τ12 = 4 s for pyrene and τ12 = 7 s for pyrene decanoic acid). This indicates that molecules like fatty acids or apolar membrane constituents must equilibrate very rapidly in a single bilayer vesicle.The water solubility or the critical micelle concentration of the probe molecules is determined and related to the transfer rates. An exchange process through the water phase via a monomeric state can be excluded.  相似文献   

7.
All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.  相似文献   

8.
On the mechanism of action of lysophospholipase-transacylase from rat lung   总被引:1,自引:0,他引:1  
Lysophospholipase-transacylase from rat lung catalyzes the transfer of palmitate from 1-palmitoyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-palmitoyl-sn-glycero-3-phosphocholine. Incorporation of palmitate into phosphatidylcholine is restricted to palmitate donated by lysophosphatidylcholine, free palmitate cannot be esterified to lysophosphatidylcholine by the enzyme. Experiments in the presence of H218O and mass spectrometric analysis of the reaction products show that 18O is incorporated into the released palmitate but not into the transesterification product phosphatidylcholine. This proofs that the hydrolytic reaction proceeds by O-acyl cleavage. Furthermore, the results strongly suggest that transfer of palmitate to lysophosphatidylcholine occurs through an intermediary covalent acyl-enzyme complex.  相似文献   

9.
Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD+ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the Km for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.  相似文献   

10.
The mode of interaction of aqueous dispersions of phospholipid vesicles is investigated. The vesicles (average diameter 950 Å) are prepared from total lipid extracts of Escherichia coli composed of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. One type of vesicle contains trans-Δ9-octadecenoate, the other type trans-Δ9-hexadecenoate as predominant acyl chain component. The vesicles show order?disorder transitions at transition temperatures, Tt = 42° C and Tt = 29° C, respectively. A mixture of these vesicles is incubated at 45° C and lipid transfer is studied as a function of time using the phase transition as an indicator. The system reveals the following properties: Lipids are transferred between the two vesicle types giving rise to a vesicle population where both lipid components are homogeneously mixed. Lipid transfer is asymmetric, i.e. trans-Δ9-hexadecenoate-containing lipid molecules appear more rapidly in the trans-Δ9-octadecenoate-containing vesicles than vice versa. At a given molar ratio of the two types of vesicles the rate of lipid transfer is independent of the total vesicle concentration. It is concluded that lipid exchange through the water phase by way of single molecules or micelles is the mode of communication of these negatively charged lipid vesicles.  相似文献   

11.
Theodore Dashman 《Life sciences》1980,27(15):1415-1422
The enol-ether amino acid, L-2-amino-4-methoxy-trans-butenoic acid (AMTB) is an inhibitor of porphobilinogen synthase (PBG synthase) when added prior to the addition of the substrate δ-aminolevulinic acid. The inhibition of PBG synthase by several stereoisomers and analogues of AMTB was investigated to determine those structural features of AMTB which may be necessary for inhibition. The D-trans isomer was also an inhibitor after preincubation, whereas the L-cis isomer inhibited with or without preincubation. The amino acid analogues, DL-vinylglycine, DL-2-aminobutanoic acid, the reduced form of L-2-amino-4-methoxy-trans-3-butenoic acid, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid and its reduced congener did not inhibit PBG synthase even with preincubation. This structure activity relationship indicates that the trans double bond and methoxy moiety of L-2-amino-4-methoxy-trans-3-butenoic acid are probably required for inhibition.Heme, when preincubated with PBG synthase, was an inactivator of the enzyme. However, when both L-2-amino-4-methoxy-trans-3-butenoic acid and heme were simulatneously preincubated with PBG synthase, inactivation of the enzyme was greater than with either compound separately. The possibility of multiple catalytic sites was suggested by the use of multiple inhibition kinetics in the presence of heme and L-2-amino-4-methoxy-trans-3-butenoic acid.  相似文献   

12.
13.
Embryos were non-surgically recovered from superovulated donors. Two trials of ipsilateral single embryo transfer were performed with the Japanese AI instrument.For the first trial, neither the AI instrument nor the cervical expander were ensheathed. The overall pregnancy rate was 33.3 % (2226). Pregnancy rates obtained from three groups with different media were almost identical.In the second trial, both the AI instrument and the cervical expander were covered with a paper sheath to minimize uterine infection. The overall pregnancy rate after the second trial was 59.1 % (1322).  相似文献   

14.
G E Pratt  S S Tobe 《Life sciences》1974,14(3):575-586
When corpora allata from adult female Schistocercagregaria are incubated invitro with either 3H-trans, trans farnesenic acid or 3H-trans, trans, cis bishomo-farnesenic acid and [methyl-14C] methionine, they fabricate large quantities of the corresponding double labelled methyl 10, 11-epoxy esters. Radio GLC of these products indicates retention of geometric configuration at the C-2 and C-6 double bonds. Separate analyses of the contents of the glands and medium after incubation show that the epoxy esters are rapidly released from the glands into the medium and that only the glands contain the corresponding unepoxidized esters. We suggest that unepoxidized esters are the intracellular intermediates in the formation of juvenile hormones from the unsaturated acids. Gel filtration shows that the epoxy esters are not released as stable protein complexes but as simple solutes into the medium. Using this method of promoting the synthesis of juvenile-hormone-active compounds, rates of biosynthesis of epoxy esters of up to 33 ng. per pair of glands per hour have been achieved.  相似文献   

15.
Behavioral comparisons of the stereoisomers of tetrahydrocannabinols   总被引:1,自引:0,他引:1  
The potencies of (?)-trans9-THC, (+)-trans9-THC, (+)-cis9-THC, (?)-trans8-THC and (+)-trans8-THC were compared in several different species. (?)-trans9-THC was 100 times more potent than (+)-trans9-THC in depressing schedule-controlled responding in monkeys. The (+)-trans isomers were less effective than their corresponding (?)-trans isomers in the dog static-ataxia test, but potency ratios could not be determined due to a lack of dose-responsiveness of the (+)-trans isomers. However, it appeared that their potency differed by at least ten fold. The potency of (+)-cis9-THC in the dog static-ataxia test was comparable to that of (+)-trans9-THC. The hypothermia in mice produced by the (?) isomers of trans9-THC and trans8-THC were 9.1 and 30.4 times greater than that produced by their respective (+)-isomers. Also, the potency ratio of the (+)- and (?)-trans9-THC was 5.6 as measured by depression of spontaneous activity in mice. The magnitude of the potency ratios of the THC stereo-isomers is dependent upon the species and the pharmacological test used.  相似文献   

16.
Liver microsomes and reconstituted cytochrome P-450 systems purified from phenobarbital or 3-methylcholanthrene pre-treated rats metabolize cyclopenta(cd)pyrene at its K-region to trans-9,10-dihydroxy-9,10-dihydrocyclopenta(cd)pyrene. The rate of formation of the K-region product is from 5% to 25% that of trans-3,4-dihydroxy-3,4-dihydro-cyclopenta(cd)pyrene. The preference of microsomes and purified cytochromes P-450 for oxygenating cyclopenta(cd)pyrene at the ethylenic C(3)–C(4) position is explainable in part by the fact that the C(4) position has the greatest electron density in the highest occupied molecular orbital.  相似文献   

17.
All-trans [11-3H]4,4-difluororetinyl acetate was synthesized by treating methyl all-trans [11-3H]4-oxoretinoate with diethylaminosulfurtrifluoride, followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol, difluororetinyl palmitate and related esters, 4-oxoretinol, 4-oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and / or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by 19F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 ± 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8–3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.  相似文献   

18.
L S Graham  L R Bennett 《Steroids》1974,23(3):311-321
Capsules prepared from polydimethylsiloxane (PDS) tubing and containing tritium labeled 11β, 17α, 21-trihydroxy-4-pregnene-3, 20-dione (cortisol) were implanted subcutaneously in six mongrel dogs. Invivo diffusion rates were found to decline at a single exponential rate after a period of rapid, initial change. Diffusion of the steroid from the capsule provided an exogenous marker which can be used for the continuous monitoring of metabolic clearance rates and production rates in the normal state and during metabolic insults. Comparison of the temporal aspects of plasma radioactive concentration and excretion of radioactivity suggests the existence of a tightly bound plasma component serving as a reservoir of cortisol.  相似文献   

19.
Following picosecond light activation, the bacteriochlorophyll and bacteriopheophytin complement of Rps.sphaeroides reaction centers depleted of ubiquinone behaves as though it has no primary electron acceptor; the excited intermediary BChlBPh2 state formed in <10 ps lasts >1 ns. Addition of ubiquinone-10 reconstitutes the very rapid electron transfer rates from the excited intermediary BChBPh state to ubiquinone; the kinetics and rate are similar to that encountered in the untreated reaction centers. Interpretation of the data presented suggests that ubiquinone is the immediate electron acceptor from BPh?. This is consistent with the model for the primary reactions leading to [(BChl)2?BPh]Q?.  相似文献   

20.
An unstable epoxide, leukotriene A4 (5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C4. In the present work synthetic leukotriene A4 was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C4 and 11-trans-leukotriene C4 were the main products with SRS activity. It was not established whether the 11-trans-compound was formed by isomerization at the leukotriene A4 or C4 stage.  相似文献   

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