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1.
A radiochemical assay for glycine N-acyltransferase activity. Some properties of the enzyme in rat and rabbit. 总被引:2,自引:1,他引:1
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We have developed a sensitive radiochemical assay of glycine N-acyltransferase activity, using phenylacetyl-CoA as the acyl donor and glycine as the acceptor. This assay measures formation of the product, phenylacetylglycine, instead of disappearance of the substrate, phenylacetyl-CoA, as did earlier assays. The subcellular location and some properties of the conjugating activity were determined in liver and kidney of the rabbit and the rat. Rabbit lung and intestine were also tested for activity. 相似文献
2.
The development of the hepatic and renal hippurate-synthesizing system, as represented by the overall reaction of the benzoyl CoA: glycine N-acyltransferase (EC 2.3.1.13) was studied in 0, 4, 8, 13, 17, 21-day and 8-week old sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase (OTC) deficiency. The enzyme system in mutant males (spf/Y) showed a retarded development in both liver and kidney cortex, which was statistically significant between 13 and 21 days of age, as compared to normal males (+/Y). Hippurate synthesis in preparations from adult (8-week old) spf/Y mice was not significantly different than the normal. Daily intraperitoneal injections of sodium benzoate in increasing concentrations (125-375 mg/kg), given between 17 and 21 days, did not cause any induction in spf/Y or +/Y mice. However, intraperitoneal sodium phenobarbital (80 mg/kg) increased the specific and total activities of the hepatic enzyme system in normal +/Y mice significantly. spf/Y tolerated a dose of 40 mg/kg only, which resulted in no significant increase of hepatic enzyme activity. The results indicate that barbiturates may induce the hippurate-synthesizing system, whereas benzoate treatment has no effect on changing its developmental profile. 相似文献
3.
K Asaoka 《The International journal of biochemistry》1991,23(4):429-434
1. In various tissues from the monkey (Macaca fuscata), acyl-coenzyme A (CoA) hydrolase activities were found to be widely distributed within a 2-10 times range and present in liver cytosol having mol. wt of ca 60,000. 2. Acyl-CoA: amino acid N-acyltransferase activity were 4-250 times higher in liver and kidney than in other tissues, even no activity in heart, lung, and plasma. 3. The transferases abounded in liver mitochondria, being distributed evenly between the intracristate space, the inner membrane, and the matrix. 4. The partially purified transferases with benzoyl-CoA or phenylacetyl-CoA as substrates were shown to have mol. wt of ca 30,000 and reacted only with glycine or L-glutamine, respectively. 5. No amino acid tested had any effects on the enzyme as either inhibitors or activators. 6. These results suggest that the enzymes that metabolize acyl-CoA constitute an alternative pathway for the excretion of nitrogen. 相似文献
4.
When bovine kidney mitochondria were assayed in the presence of Triton X-100, they were found to contain glycine N-acyltransferase activity toward the CoA-adducts of benzoate, butyrate, isovalerate, naphthylacetate, phenylacetate, and salicylate. Heptanoyl-CoA activity was masked by high acyl-CoA hydrolase activity. All activities found in detergent-lysed mitochondria, and also that toward heptanoyl-CoA, could be released in soluble form by repeated cycles of freeze-thawing. Activity in the particle-free lysate decreased in the order: phenylacetyl-CoA >benzoyl-CoA >salicylyl-CoA >butyryl-CoA >naphthylacetyl-CoA >heptanoyl-CoA >isovaleryl-CoA. This is quite different from liver, where the activity toward the arylacetic acids is much lower and the other activities are higher. This reflects a major difference in the relative expression of the aralkyl and arylacetyl transferases between liver and kidney. The phenylacetyl-CoA and naphthylacetyl-CoA activity purified with a single protein which is termed the arylacetyl transferase. This enzyme was similar to the hepatic arylacetyl transferase in terms of its sensitivity to sulfhydryl reagents, response to cations, and molecular weight (33,500). Activity toward benzoyl-CoA also purified as a single form which was similar to the hepatic form in its molecular weight (34,000), response to cations, and kinetic properties. Conditions leading to the inhibition of this kidney form and also the hepatic form by p-mercuribenzoate are described. 相似文献
5.
Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver. 相似文献
6.
A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens. 相似文献
7.
A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.
J B Kwakye M R Johnson S Barnes R B Diasio 《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,100(1):131-136
1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers. 相似文献
8.
Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol 总被引:3,自引:0,他引:3
R Chanderbhan T Tanaka J F Strauss D Irwin B J Noland T J Scallen G V Vahouny 《Biochemical and biophysical research communications》1983,117(3):702-709
Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity. 相似文献
9.
M Razanoelina N Freund J Bismuth J P Geloso E Delaval 《Journal of developmental physiology》1991,16(5):283-286
Oxygen consumption (VO2) and beta-hydroxyacyl-CoA dehydrogenase (beta OAC) activity were measured in isolated mitochondria of developing rat kidney from late fetal to adult age. In the presence of palmitoyl-L-carnitine, VO2 consumption was higher in suckling than in adult rats while beta OAC activity rose during the postnatal period and declined after weaning. During postnatal development, the high level of mitochondrial fatty acid oxidation was linked to the high level of fatty acid supply and any change in lipid diet altered mitochondrial fatty acid oxidation. By contrast at adult age, a high fat diet did not change either mitochondrial fatty acid oxidation or beta OAC activity measured in two nephron structures (PCT and mTAL). Dietary lipids seem to play an important role in the evolution of mitochondrial fatty acid oxidation in developing rat kidney. 相似文献
10.
Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases 总被引:4,自引:0,他引:4
D J Carothers C Raefsky-Estrin G Pons M S Patel 《Archives of biochemistry and biophysics》1987,256(2):597-605
We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system. 相似文献
11.
Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in rat liver 总被引:2,自引:0,他引:2
P G Killenberg 《Journal of lipid research》1978,19(1):24-31
An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid N-acyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions. Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. Choloyl-CoA synthetase is associated with the microsomal membranes and bile acid-CoA:amino acid N-acyltransferase activity with the postmicrosomal supernatant. No significant amino acid N-acyltransferase activity is present in the lysosome fraction. These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids. 相似文献
12.
Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.
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K P Wong 《The Biochemical journal》1976,158(1):33-37
The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals. 相似文献
13.
F Trejtnar O Jindrová H Lisková J Siroká J Kv?tina 《Physiological research / Academia Scientiarum Bohemoslovaca》1991,40(1):81-86
The enzymatic activity of the mixed-function oxidase system in the kidney and liver was evaluated by means of an in vitro N-demethylation activity assay with aminopyrine as the substrate. Renal and hepatic demethylation activity of 9000 x g supernatant fraction was determined in the rat, rabbit, and guinea-pig. In terms of interspecies comparison, the renal tissue demethylation activities were on a similar level with a slight increase in the order guinea-pig, rabbit and rat. In relation to hepatic activity, these relative demethylation activities of renal tissue had the same values in the rat and rabbit, whereas that in the guinea pig was significantly lower. The distribution of demethylation activity in the kidney was determined by comparing the cortex and medullary activity in relation to the total kidney tissue activity in the rabbit and guinea-pig. Although the higher demethylation activities were obtained in rabbit renal preparations and low demethylation activity was detected in the guinea-pig renal medulla only, no significant interspecies differences were found by the statistical evaluation. It may be concluded that the mixed-function oxidase system responsible for renal demethylation activity seems to be concentrated in the renal cortex and its distribution coincides in the rabbit and guinea-pig kidney. 相似文献
14.
The development of the glycine cleavage system was studied in liver mitochondrial extracts from neonatal and adult rats. The enzyme activity in 2-day-old pups was 29.3% of that measured in the adult and was found to increase in an age-dependent manner. Measurement of hepatic free amino acid concentrations at the neonatal ages showed that glycine levels were highest at 2 days and at 14 days were about 48% of those in the 2-day-old pups. Serine levels did not change between the age of 2 and 14 days. A developmental delay in the glycine cleavage system is responsible for the high levels of glycine in the neonatal rat liver. 相似文献
15.
van der Westhuizen FH Pretorius PJ Erasmus E 《Journal of biochemical and molecular toxicology》2000,14(2):102-109
The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs. 相似文献
16.
Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver 总被引:2,自引:0,他引:2
An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine. 相似文献
17.
L Zychlinski M R Montgomery 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1987,86(2):325-328
Pulmonary mitochondrial monoamine oxidase (MAO) activity was examined in preparations from rat, rabbit and guinea-pig with 12 different amines as substrates: serotonin, norepinephrine, and octopamine (type A specific); tryptamine, benzylamine, 5-methoxytryptamine, 5-methyltryptamine, p-methoxyphenylethylamine, and 3,4-dimethoxyphenethylamine (type B specific); and tyramine, dopamine and 3-methoxytyramine (type A + B specific). The oxidation of type A and type A + B substrates was greater in guinea-pig lung mitochondria than in rat or rabbit preparations. Except for benzylamine, the oxidation of type B substrates was similar in all three species. Benzylamine was not oxidized by guinea-pig lung mitochondria but was actively metabolized by rat and rabbit preparations. 相似文献
18.
Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT. 相似文献
19.
Catherine Brégère Igor Rebrin Timothy K. Gallaher Rajindar S. Sohal 《Free radical biology & medicine》2010,48(4):609-618
This study examined the protein targets of nitration and the consequent impact on protein function in rat kidney mitochondria at 4, 13, 19, and 24 months of age. Succinyl-CoA transferase (SCOT), a rate-limiting enzyme in the degradation of ketone bodies, was the most intensely reactive protein against anti-3-nitrotyrosine antibody in rat kidney mitochondria. However, subsequent mass spectrometric and amino acid analyses of purified SCOT indicated that tryptophan 372, rather than a tyrosine residue, was the actual site of simultaneous additions of nitro and hydroxy groups. This finding suggests that identification of nitrated tyrosine residues based solely on reactivity with anti-3-nitrotyrosine antibody can be potentially misleading. Between 4 and 24 months of age, the amounts of SCOT protein and catalytic activity, expressed per milligram of mitochondrial proteins, decreased by 55 and 45%, respectively. SCOT, and particularly its nitrated carboxy-terminal region, was relatively more susceptible to in vitro proteolysis than other randomly selected kidney mitochondrial proteins. The age-related decreases in SCOT protein amount and catalytic activity were prevented by a relatively long-term 40% reduction in the amount of food intake. Loss of SCOT protein in the aged rats may attenuate the capacity of kidney mitochondria to utilize ketone bodies for energy production. 相似文献
20.
Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver 总被引:2,自引:0,他引:2
M R Johnson S Barnes J B Kwakye R B Diasio 《The Journal of biological chemistry》1991,266(16):10227-10233
The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver. 相似文献