Purification and kinetic properties of ox brain histamine N-methyltransferase.
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ABSTRACT:
SUBMITTER: Gitomer WL
PROVIDER: S-EPMC1153084 | biostudies-other | 1986 Feb
REPOSITORIES: biostudies-other
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Project description:The substrate kinetic properties of histamine methyltransferase from human skin were studied at limiting concentrations of both histamine and S-adenosylmethionine. Substrate inhibition by histamine was observed at concentrations above 10 microM. Primary plots showed evidence of a sequential reaction mechanism. The Michaelis constants were derived from secondary plots of slopes from the primary plots ([S]/v versus [S]) versus reciprocal of the second substrate concentration. The mean Km values for histamine and S-adenosylmethionine were 4.2 and 1.8 microM respectively. Histamine in concentrations of 25-100 microM inhibited enzyme activity uncompetitively with respect to S-adenosylmethionine. No substrate inhibition was observed with S-adenosylmethionine. To elucidate the reaction mechanism further, inhibition by the two products, S-adenosylhomocysteine and 1-methylhistamine, was studied. S-Adenosylhomocysteine inhibited non-competitively with respect to histamine and competitively with respect to S-adenosylmethionine. 1-Methylhistamine inhibited non-competitively with respect to histamine and to S-adenosylmethionine. These results are interpreted as providing evidence for an ordered sequential Bi Bi reaction mechanism, with the methyl-group donor S-adenosylmethionine as the first substrate that adds to the enzyme and histamine as the second substrate. 1-Methylhistamine is the first product to leave the enzyme and S-adenosylhomocysteine is the second. The results are discussed in terms of the possible role that this enzyme could play in the modulation of histamine-mediated reactions in skin.
| S-EPMC1162467 | biostudies-other
Project description:Brain histamine is a neurotransmitter and regulates diverse physiological functions. Previous studies have shown the involvement of histamine depletion in several neurological disorders, indicating the importance of drug development targeting the brain histamine system. Histamine N-methyltransferase (HNMT) is a histamine-metabolising enzyme expressed in the brain. Although pharmacological studies using HNMT inhibitors have been conducted to reveal the direct involvement of HNMT in brain functions, HNMT inhibitors with high specificity and sufficient blood?brain barrier permeability have not been available until now. Recently, we have phenotyped Hnmt-deficient mice to elucidate the importance of HNMT in the central nervous system. Hnmt disruption resulted in a robust increase in brain histamine concentration, demonstrating the essential role of HNMT in the brain histamine system. Clinical studies have suggested that single nucleotide polymorphisms of the human HNMT gene are associated with several brain disorders such as Parkinson's disease and attention deficit hyperactivity disorder. Postmortem studies also have indicated that HNMT expression is altered in human brain diseases. These findings emphasise that an increase in brain histamine levels by novel HNMT inhibitors could contribute to the improvement of brain disorders.
| S-EPMC6386932 | biostudies-literature
Project description:BACKGROUND:Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes. RESULTS:Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K(M) for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25 degrees C-45 degrees C in vitro. Only at a temperature of 50 degrees C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme. CONCLUSIONS:HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K(M) values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.
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Project description:The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.
| S-EPMC1177436 | biostudies-other
Project description:The enzyme S-adenosylmethionine (AdoMet): myelin basic protein (MBP) methyltransferase was purified 250-fold from bovine brain with an overall yield of 130%, relative to crude supernatant. The purification involves acid-base and (NH4)2SO4 precipitation, chromatography over Sephadex G-100 and DEAE-cellulose, followed by preparative isoelectric focusing. The enzyme has a pI of 5.60 +/- 0.05, and the Mr is estimated to be between 71,000 (from SDS/polyacrylamide-gel electrophoresis) and 74,500 (from gel filtration). The enzyme is stable at 37 degrees C for over 2 h, is stable frozen and does not require metal ions or reductants. The enzyme shows a high specificity for MBP and does not accept polyarginine as a substrate; F1 histone is methylated at 37% of the rate of MBP. Methylation occurs on an arginine residue in a single h.p.l.c.-resolvable peptide from the tryptic cleavage of MBP. Simple saturation kinetics are observed with respect to both substrates, with Km values of 18 microM and 32 microM for MBP and AdoMet respectively. The simplest kinetic mechanism that is consistent with the data requires ordered rapid-equilibrium binding, with AdoMet as the first substrate. The enzyme isolated in this work is different, both physically and kinetically, from the histone-specific arginine methyltransferases described by other workers. A new, simple, assay system for the methylation of MBP is described.
| S-EPMC1148836 | biostudies-other
Project description:1. Two soluble arylsulphatases (A and B) have been extracted from ox brain by a modified Albers autolysis method and purified by acetone and ammonium sulphate precipitation and dialysis. 2. A 1600-fold purification was achieved with arylsulphatase A and 320-fold purification with arylsulphatase B. 3. The specific activity of arylsulphatase A was 266000 4-nitrocatechol units/mg. of protein N, and that of arylsulphatase B was 64600units/mg. of protein N. 4. Arylsulphatase A seems to be electrophoretically homogeneous. 5. With 3mm-dipotassium 2-hydroxy-5-nitrophenyl sulphate as substrate the optimum pH for the activity of arylsulphatase A was 4.7, and for arylsulphatase B it was 6.1 with a 60mm solution of the same substrate.
| S-EPMC1264648 | biostudies-other
Project description:The binding affinity of ligands for their receptors is determined by their kinetic and thermodynamic binding properties. Kinetic analyses of the rate constants of association and dissociation (kon and koff, respectively) of antihistamines have suggested that second-generation antihistamines have a long duration of action owing to the long residence time (1/koff) at the H1 receptors. In this study, we examined the relationship between the kinetic and thermodynamic binding properties of antihistamines, followed by an evaluation of the structural determinants responsible for their kinetic binding properties using quantitative structure-activity relationship (QSAR) analyses. We found that whereas the binding enthalpy and entropy might contribute to the increase and decrease, respectively, in the koff values, there was no significant relationship with the kon values. QSAR analyses indicated that kon and koff values could be determined by the descriptors FASA_H (water-accessible surface area of all hydrophobic atoms divided by total water-accessible surface area) and vsurf_CW2 (a 3D molecular field descriptor weighted by capacity factor 2, the ratio of the hydrophilic surface to the total molecular surface), respectively. These findings provide further insight into the mechanisms by which the kinetic binding properties of antihistamines are regulated by their thermodynamic binding forces and physicochemical properties.
| S-EPMC7957501 | biostudies-literature
Project description:Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.
| S-EPMC1144882 | biostudies-other
Project description:1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.
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Project description:Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.
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