Human Platelet Monoamine Oxidase Research Articles

Substrate specificity of human platelet monoamine oxidase B activity Parkinson's and Alzheimer's disease.

Substrate specificity of human platelet monoamine oxidase B activity Parkinson's and Alzheimer's disease.

Inhibition of monoamine oxidases by haloperidol and its metabolites: pharmacological implications for the chemotherapy of schizophrenia.

The effect of haloperidol and its metabolites on human platelet monoamine oxidase B (MAO-B) and human placenta monoamine oxidase A (MAO-A) in vitro has been investigated. We found that 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-pyridinium (HP+), 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HTP) and 4-chlorophenyl-1,2,3,6-tetrahydropyridine (CPTP) are potent inhibitors of MAO. HP+ appeared to be a reversible, uncompetitive and selective MAO-B inhibitor with a Ki of 0.83 microM. HTP was found to be an irreversible, uncompetitive and selective MAO-B inhibitor (Ki of 1.84 microM). CPTP inhibits both MAO-A and MAO-B. Some other haloperidol metabolites, i.e. 4-(4-chlorophenyl)-4-hydroxypyridine (CPHP), 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine N-oxide (HTPNO) and reduced haloperidol (RHAL), do not inhibit MAO to any appreciable degree at concentrations up to 100 microM. The results suggest that haloperidol metabolites may contribute to the reduction of platelet MAO-B activity in schizophrenic patients undergoing neuroleptic chemotherapy. An examination of the literature reveals that schizophrenic patients with low platelet MAO activity exhibit a strong association with the use of haloperidol. Other possible pharmacological implications of the inhibition of MAO activity are discussed.

Determination of platelet monoamine oxidase activity by high-performance liquid chromatography with electrochemical detection

A procedure for determining human platelet monoamine oxidase (MAO) with dopamine (DA) as substrate is described. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used to separate and detect components of the reaction mixture. The method for platelet preparation was also improved and only 2 ml of blood were required. Following a 10-min incubation of the platelet preparation with DA in 0.1 M Tris buffer (pH 9.0), excess DA substrate was removed by adsorption on a cation-exchange resin. The reaction product, 3,4-dihydroxyphenylacetaldehyde, was adsorbed on acid-washed alumina, eluted with 0.1 M perchloric acid and analyzed by HPLC. Simple, clean chromatograms were obtained with good reproducibility using 3,4-dihydroxybenzylamine as an internal standard. The within-sample, between-samples and between-day relative standard deviations were 0.9, 3.7 and 6.1%, respectively. The apparent Michaelis constant and maximum velocity were 0.10 m M and 0.37 nmol/min · mg protein, respectively. This HPLC-ED method offers a good alternative to methods using radioactivity.

Modification of substrate-inhibitor affinities of human platelet monoamine oxidase B in vitro

The rate of benzylamine utilization by monoamine oxidase (MAO)-B from human blood platelets was 2-4 times higher than that for octopamine. Both activities were inhibited 100% by 10(-7) M deprenyl (a specific MAO-B inhibitor) and were not affected by clorgyline (a specific MAO-A inhibitor) or by polyclonal antibodies to MAO-A. The preincubation of platelet MAO-B with purified MAO-A from mitochondrial membranes of human placenta resulted in appearance of excess octopamine activity. This additional activity was not precipitated by antibodies to MAO-A or inhibited by deprenyl but was inhibited by clorgyline. Incubation of the MAO-A preparation from placenta at 45 degrees C for 15 min before its preincubation with MAO-B caused 50% loss of both activities. Protease inhibitors had no effect on the modification of MAO. These data indicate that MAO-A or a factor tightly bound to it can modify MAO-B yielding a form of the enzyme with both MAO-A and MAO-B substrate and inhibitor affinities and MAO-B immunospecificity.

Formula omitted] inhibition of human platelet monoamine oxidase by phenothiazine derivatives

Nineteen phenothiazines were tested for in vitro inhibition of human platelet type B monoamine oxidase (MAO). The inhibition potency was highly dependent on structures of their side chains. The inhibition was most potent for drugs with (hydroxyethyl-piperazinyl)propyl chains followed in decreasing order by those with (N-methylpiperazinyl)propyl, (2-dimethylamino-2-methyl)ethyl and 3-dimethylaminopropyl chains. Kinetic analyses were carried out for promazine, promethazine, perazine and perphenazine as representatives of each group; the four drugs showed competitive inhibition, and K i values of 124, 31.4, 19.2 and 22.6 μM, respectively.

Determination of platelet monoamine oxidase by new continuous spectrophotometric method.

A simple, continuous spectrophotometric method for the determination of tissue monoamine oxidase based on the oxidation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) using peroxidase has already been described (Ivanović, I. & Majkić-Singh, N. (1986) Fresenius Z. Anal. Chem. 324, 307). In the present study the method is optimised for platelet monoamine oxidase assay and applied to healthy persons and schizophrenic patients. The obtained data were statistically analysed. The continuous ABTS method is sensitive, precise (CV below 6.9%) and linear up to 83 U/g protein. Comparison with the end-point method of Szutowicz et al. (1984) Anal. Biochem. 138, 86-94) gave a good correlation (r = 0.983). The reference values for the activity of human platelet monoamine oxidase by the new continuous ABTS method are 25 to 42 U/g protein (means = 33.2 U/g protein, CV = 15.5%, n = 67). No differences were found between females and males, or between three age groups ranging from 21 to 52 years. The patients with chronic (n = 76) or acute (n = 17) schizophrenia had significantly lower monoamine oxidase activities compared with normal values (p less than 0.005), which indicates that platelet monoamine oxidase can be a possible marker for schizophrenic diseases.

Human monoamine oxidase. Lack of brain and platelet correlation.

Monoamine oxidase (MAO) exists in two forms, MAO A and MAO B. Both are present in human brain, but the human platelet contains only MAO B. We studied whether individual variations in the activity of human platelet MAO B reflect individual variations in cerebral cortical MAO activities. Optimal conditions were determined for the measurement of MAO activities in both the platelet and cerebral cortex, obtained from 14 patients with epilepsy during clinically indicated neurosurgery. There was no significant correlation between the activities of MAO B in the cerebral cortex and platelets of these patients. Platelet MAO B activities also failed to correlate significantly with cerebral cortical MAO A activities. However, there was a significant positive correlation between cerebral cortical MAO A and MAO B activities. Individual variations in platelet MAO B activities do not reflect individual variations in either cerebral cortical MAO B or MAO A activities in patients with epilepsy who undergo neurosurgery.

Conversion of the neurotoxic precursor 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine into its pyridinium metabolite by human platelet monoamine oxidase type B

MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) causes selective and irreversible degeneration of the substantia nigra of human and non-human primates. In the central nervous system, the oxidative metabolism of MPTP to 1-methyl-4-phenyl-pyridinium (MPP +) by monoamine oxidase type B (MAO-B) seems to be a critical feature in the neurotoxic process. We now report that [ 3H]MPTP is rapidly converted in vitro into [ 3H]MPP − by human platelet MAO-B. The formation of [ 3H]MPP + in human platelets is prevented by specific MAO-B but not by MAO-A or by 5-hydroxytryptamine uptake inhibitors.

Effect of pregnancy on human platelet monoamine oxidase and phenolsulphotransferase M and P

SummaryThere were no significant changes in platelet phenolsulphotransferase M and monoamine oxidase activities in pregnant women compared with controls and women postpartum, although mean monoamine oxidase values were about 10 per cent higher during pregnancy. Platelet phenosulphotransferase P activity tended to decline during pregnancy and rose substantially postpartum. Mean values postpartum were higher than those in the pregnant group as a whole (P > 0.01) and in women during the last trimester of pregnancy (P > 0.01). If these platelet findings reflect changes elsewhere in the body, they might imply that women towards the end of pregnancy are more sensitive and women postpartum less sensitive to the action of drugs that are inactivated by sulphoconjugation.

Activation of monoamine oxidase by high molecular weight fractions of human plasma.

The activation of human platelet monoamine oxidase (MAO) and rat brain mitochondrial MAO ( RBM -MAO) by human plasma were studied. The deamination of two different substrates, tyramine and phenethylamine (PEA) was investigated. The increase in MAO activity in the presence of human plasma can be explained by the observed decrease in the apparent Km for the amine (tyramine, PEA). This activation pattern was the same both for human platelet MAO and RBM -MAO. The activating properties of human plasma were recovered in high molecular weight fractions after gel filtration.

Purification of Human Platelet Monoamine Oxidase B by High Performance Liquid Chromatography

Abstract Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chromatography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0–1%) of octyl-β-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently labeled with [3H]-pargyline [3H(G)] or catalytic activity using [14C-methylene]-benzylamine as substrate. [3H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit Mr (approximately 59,000) of MAO B were determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.

Solubilization of monoamine oxidase by octylglucoside and Triton X-100

The suitability of octyl-β- d-glucoside as a solubilizing agent for human platelet monoamine oxidase from the outer mitochondrial membrane was investigated and compared to Triton X-100. The properties measured were the effect of the detergents on storage, stability, activity measurements, and solubility of monoamine oxidase, which was measured by pargyline titration. Comparisons on the types of proteins solubilized were also determined by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. Our results demonstrated that octyl-β- d-glucoside was equivalent to purified Triton X-100 and is probably the better detergent for enzyme characterization because of its well-defined chemical composition and its ease of removal by dialysis.

Human platelet monoamine oxidase—A useful enzyme in the study of psychiatric disorders?

Human platelet monoamine oxidase—A useful enzyme in the study of psychiatric disorders?

Monoamine oxidase in human platelets: Kinetics and methodological aspects

The kinetic properties of human platelet monoamine oxidase (MAO) were examined in 20 apparently healthy controls. The mean value (±S.D.) of the maximum velocity ( V) was found to be 5.36 ± 1.97 pmoles of product formed/10° platelets/min and the Michaelis-Menten constants were for phenethylamine ( K PEA m ) 14.6 ± 8.20 μM and for oxygen ( K m O 2 )254 ± 125 μM, when assayed in 0.1 M phosphate buffer, pH 7.4. The relation between the value of the corresponding apparent constants was studied. Inhibition of the enzyme activity was seen at 20 μM of PEA and 180 μM of oxygen. The enzyme kinetics were also studied at different pH. Two p K values were found, p K 1 = 6.65 and p K 2 = 6.95. The influence of homogenization on the MAO activity was compared with the activity in the undisrupted platelet. At PEA concentrations below 10 μM higher MAO activities were found in the intact cell. A 15 per cent loss of activity was detected in platelet samples after storing at −20° for three and a half years.

Human platelet monoamine oxidase activity in health and disease: a review.

The most readily available source of monoamine oxidase in man is the platelet, although only the B form of the enzyme is represented in this site. Platelet activity is higher in women than in men. The enzyme activity is generally stable and is partly under genetic control. There is some evidence that individuals with low activity have a higher psychiatric morbidity than those with high activity. Despite some negative studies, the consensus of publication dealing with schizophrenia, migraine, and alcoholism find that mean platelet monoamine oxidase activity in the patient group is lower than in the controls. Values are raised in unipolar depression. Technical differences, or patient or control group heterogeneity, might well account for the absence of unanimity in the literature. A considerable degree of overlap between patient and control values, whatever the clinical diagnosis, appears to be the standard finding. Apart from these neuropsychiatric disturbances, platelet monoamine oxidase activity is raised in megaloblastic anaemia and reduced in iron deficiency anaemia. Although altered enzyme activity values may be linked to abnormal platelet populations in some of the haematological disorders discussed, in general the causes of abnormal platelet monoamine oxidase activity are unknown.

Molecular weight differences between human platelet and placental monoamine oxidase

The molecular weights of the active site subunits of human placental and platelet monoamine oxidase have been compared. These tissues were chosen as each appears to contain only one form of the enzyme, type A in placenta and type B in platelets. We found that the biochemical properties (substrate affinity and inhibitor sensitivity) clearly distinguish between the enzymes from these two sources, whether in the membrane-bound or detergent-solubilized state. The suggestion that such differences result from the existence of distinct molecular forms was strengthened by the observation that the active site subunits labelled specifically with [ 3H]pargyline differ in their apparent molecular weights and in the electrophoretic pattern of their partial proteolytic digest products.

Molecular characteristics of human platelet monoamine oxidase.

The monoamine oxidase B (MAO-B) selective inhibitor J-508 (N-methyl-N-propargyl-(1-indanyl)-ammonium chloride) appears to interact with MAO-B in a manner consistent with a "suicide" reaction. Because of this property, J-508 could be used, under the appropriate conditions, to "titrate" the concentration of MAO-B active centres in the human platelet, although some non-specific binding of this compound to sites other than the active centre of this enzyme form was found, thus limiting the accuracy of the titration method. The molecular characteristics of human platelet MAO-B (Km, Vmax, approximate enzyme concentrations and molecular turnover members) towards three of its monoamine substrates have been estimated. The natural variation of platelet MAO-B activity from individual to individual is due to a variation in the Vmax without a variation in the Km towards benzylamine is substrate, and is based, at least in part, upon a variation in the concentration of this enzyme form.

The interaction between human platelet monoamine oxidase, its monoamine substrates and oxygen

The interaction of human platelet MAO-B with three substrates (β-phenethylamine, tryptamine and benzylamine) has been investigated in an attempt to determine whether or not there exists heterogeneity of this enzyme form. Treatment with pargyline, thermal denaturation and 2-butanone affected the enzyme activity to the same degree, regardless of the amine substrate used. Mixed substrate experiments indicated that the substrates inhibited each other in a competitive manner with K i values close to their K m values. The activity of MAO-B was increased in an uncompetitive manner as the oxygen concentration was raised. However, the degree of this increase was dependent upon the amine substrate used to assay for activity. These results are consistent for an enzyme with a single binding site for amine substrates and a possible multiplicity of binding sites for oxygen.

Effect of temperature on human platelet monoamine oxidase

Effect of temperature on human platelet MAO activity was studied by using three different substrates at a temperature range of 12–44°C. The Arrhenius plot of ln V max versus 1 T yielded a straight line when tryptamine was used as substrate. On the other hand, a nonlinear relationship was observed with both benzylamine and PEA as substrates. This suggests that there are multiple catalytic sites or multiple forms in human platelet MAO.

Determination of platelet monoamine oxidase activity in human platelet-rich plasma— A new microfluorescent assay utilizing kynuramine as substrate

A simple assay system for the measurement of human platelet monoamine oxidase is described. The assay is based on the fluorometric measurement of 4-hydroxyquinoline produced by enzymatic oxidative deamination of kynuramine. The results obtained on 21 individualscorrelate ( r 2 = 0.97) with data obtained in parallel with a [ 14C]benzylamine/pargyline assay. This assay system appears to be technically and economically favorable as a method for screening large numbers of individuals in order to establish distributions of monoamine oxidase activities for normal and affected populations.