Perinatal development of, and effect of chemical pretreatment on, glycine N-acyltransferase activities in liver and kidney of rabbit and rat.
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ABSTRACT:
SUBMITTER: James MO
PROVIDER: S-EPMC1185695 | biostudies-other | 1978 May
REPOSITORIES: biostudies-other
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Project description:Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.
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Project description: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.
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Project description:The activities of two phosphate-dependent glutaminase reactions characteristic of adult rat liver and kidney were determined in these organs from 2(1/2) days before to 7 days after birth and compared with the activities in the adult tissues. In the kidney, before and after birth, only the kidney type of activity was detected, and it increased in concentration in parallel with the steady growth of that organ throughout the period examined. In the liver, however, the kidney type of activity was the only one present 2(1/2) days before birth, and its concentration decreased to barely significant values by the end of the first week after birth. In contrast, the liver type of activity appeared only just before birth and increased to 60% of adult values over the next 4 days. There was no obvious relation between these changes in glutaminase type and changes in liver weight, protein content and total cell number that occurred during this time. But there was a very close correlation between the fall in kidney-type activity and the estimated fall in haematopoietic-cell number in liver.
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Project description:The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s0.5,benz = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.
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