Creed W. Abell

Immunocytochemical localization of monoamine oxidases A and B in human peripheral tissues and brain.

Monoamine oxidases (MAO; EC 1.4.3.4.) A and B occur in the outer mitochondrial membrane and oxidize a number of important biogenic and xenobiotic amines. Monoclonal antibodies specific for human MAO A or B and immunocytochemical techniques were used to visualize the respective enzymes in human placenta, platelets, lymphocytes, liver, brain, and a human hepatoma cell line. MAO A was observed in the syncytiotrophoblast layer of term placenta, liver, and a subset of neurons in brain, but was not observed in platelets or lymphocytes, which are known to lack type A enzyme. MAO B was observed in platelets, lymphocytes, and liver, but not in placenta, which contains little or no MAO B. MAO B was also observed in a subset of neurons in the brain that was distinct from that which contained MAO A. MAO A and MAO B were also observed in some glia. Unlike most tissues examined, liver cells appeared to contain both forms of the enzyme. These studies show that MAO A and MAO B can be specifically visualized by immunocytochemical means in a variety of human cells and tissues and can provide a graphic demonstration of the high degree of cell specificity of expression of the two forms of the enzyme.

Metabolism of the neurotoxin in MPTP by human liver monoamine oxidase B

The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was oxidized to dihydropyridine MPDP+ and pyridine MPP+ by preparations of monoamine oxidase B (MAO B), including pure human liver MAO B:monoclonal antibody complex. K m,app values for MPTP and benzylamine, a preferred MAO B substrate, were 316 and 64 μM, respectively. 4‐Phenyl‐1,2,3,6‐tetrahydropyridine (PTP), the nor derivative of MPTP, was also a substrate (K m,app = 221μM). MPDP+ MPTP, and MPP+, but not PTP, were found tobe irreversible inhibitors of MAO B. Our studies support the hypothesis that MPTP is oxidized in primate brain by MAO B to MPDP+ which is then converted to MPP+ a major metabolite found in the substantia nigra.

Serotonergic conversion of MPTP and dopaminergic accumulation of MPP

[3H]MPP+ had lower K m and higher V max values for its accumulation in rat brain synaptosomes than did [3H]MPTP. The kinetic parameters favored the uptake of [3H]MPP+ in the striatum to that in hypothalamus, whereas they were equally favorable for the uptake of [3H]MPTP in both regions. Hypothalamic uptake of [3H]MPTP and [3H]MPP+ was inhibited by desipramine, imipramine, norepinephrine, and serotonin. Striatal uptake of [3H]MPP+ and [3H]MPTP was blocked by nomifensine and dopamine. These results support the concept that MPTP accumulates in serotonergic neurons where it is oxidized by monoamine oxidase B to MPP+, which is released and then is selectively accumulated in dopaminergic neurons via the dopamine uptake system.

Further insight into the mode of action of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Chemical reactions of MPDP+, a recognized intermediate in the metabolic conversion of the neurotoxin MPTP by monoamine oxidase B into its major metabolite MPP+ were studied.Addition of cyanide to MPDP+ bromide in aqueous solutions afforded cyano‐compound 5 which isomerized in the presence of silica gel into compound 6. Both 5 and 6 when heated yielded a third isomer 7. MPDP+ bromide disproportionated into MPTP and MPP+ in aqueous solution near neutral or slightly alkaline pH, a reaction which also occurred when MPDP+ bromide was treated with an amine in dichloromethane solution. Disproportionation of MPDP+ at physiological pH may be of biochemical significance, since formation of MPP+ from MPDP+ can occur non‐enzymatically. MPTP, MPDP+, and MPP+ inhibited dopamine uptake in rat synaptosomal preparations with I50 values of 30, 37, and 3.4 μM, respectively. The competition of these compounds with dopamine for uptake sites in the membrane may contribute in part to the reduced levels of dopamine observed in animals treated with MPTP.

Platelet MAO concentration and molecular activity: II. Comparison of normal and schizophrenic populations

Platelet monoamine oxidase (MAO B) in 59 normal and 57 RDC-diagnosed medicated and unmedicated schizophrenic subjects was analyzed for whole platelet and extracted activities, specific concentration, and molecular activity. A novel radioimmunoassay using a monoclonal antibody elicited to human platelet MAO was used. Female schizophrenics showed no differences from female normals in MAO measures; however, these data could not be clearly evaluated because of confounding effects of age and drugs. Male schizophrenics treated with neuroleptics expressed significantly reduced whole platelet MAO activity, compared to untreated male patients. Compared with normal males, male schizophrenics showed significantly lowered molecular activities, along with elevated specific concentrations, which did not appear to be explained solely by drug usage. Additional mechanisms explaining the diminished molecular activity in male schizophrenics may be the presence of an endogenous irreversible inhibitor or a genetically determined, possibly structural, variant of MAO B.

Isolation of pure, catalytically active human liver monoamine oxidase B: Antibody complex

Monoamine oxidase B was purified from human liver mitochondria using a monoclonal antibody, MAO B-1C2, which recognizes monoamine oxidase B but not A. Triton X-100 extracts of mitochondria were incubated with purified MAO B-1C2 (IgG1), and the catalytically active enzyme:antibody complex was isolated by affinity chromatography on Protein A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex revealed the presence of four polypeptide bands (monoamine oxidase B, 57,900 dalton; antibody heavy chain, 52,200 dalton; and two light chains, 29,400 and 27,700 dalton), and indicated a 1:1 stoichiometric ratio of enzyme to antibody. This method gave 154-fold purification of the enzyme from mitochondria.

An enzymatic assay of plasma phenylalanine and tyrosine for the detection and management of phenylketonuria

Abstract An enzymatic method for the determination of plasma phenylalanine (Phe) and tyrosine (Tyr) in newborns and phenylketonurics is described. The method uses yeast phenylalanine ammonia-lyase to catalyze the conversion of l -Phe to transcinnamic acid and l -Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. This method, requiring only one drop of blood, is simple, quantitative, and time efficient, and it has been adopted for the management of phenylketonuria (PKU). It can be used for monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, for diagnosis of classical PKU through the Phe-loading test, and for detection of PKU heterozygotes. A total of 350 plasma samples collected from 31 PKU patients over a 4 year period were analyzed and yielded results similar to those obtained by automated amino acid analysis. In an exploratory study, 1084 newborn infants were screened for PKU by our enzymatic method and the Guthrie test. In this group we detected 5 infants with Phe levels greater than 8 mg/dl plasma and 2 infants with Tyr levels greater than 8 mg/dl plasma, while the Guthrie test on the same subjects gave no positives.

Platelet MAO concentration and molecular activity: I. New methods using an MAO B-specific monoclonal antibody in a radioimmunoassay

New methods for determination of specific concentration and molecular activity of monoamine oxidase (MAO) in platelets are described and evaluated in parallel with specific activity measures, performed in whole platelets and platelet extracts. Platelet MAO specific concentration is determined in platelet extracts by a radioimmunoassay, using a monoclonal antibody that recognizes human MAO B, the form that occurs in platelets, but not MAO A. All four platelet MAO measures are found to be reliable and stable, and thus are suitable for long-term comparisons of normal and clinical populations, such as those reported in Part II of this report. The new measures of enzyme concentration and molecular activity make available important information about the state of MAO B molecules in a given individual that reflects the genetic expression and control of the enzyme.

MPTP metabolites inhibit rat brain glutathione S-transferases

1-Methyl-4-phenyl-2,3-dihydropyridinium and 1-methyl-4-phenyl-pyridinium species, metabolites of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, non-competitively inhibit glutathione S-transferases of rat brain in vitro. The Ki values for 1-methyl-4-phenyl-2,3-dihydropyridinium bromide and 1-methyl-4-phenyl-pyridinium bromide are 0.67 and 0.3 mM, respectively. Inhibition of these enzymes may lead to impairment of cellular defense mechanisms.

Sodium periodate stimulation of human lymphocytes: A comparison between normal and chronic lymphocytic leukemia lymphocytes

Abstract Lymphocytes from healthy donors and from patients with chronic lymphocytic leukemia (CLL) were stimulated to divide with sodium periodate. The time of maximal response of normal lymphocytes to sodium periodate (NaIO 4 ) was earlier than that observed to phytohemagglutinin (PHA), but the magnitude was lower. In comparison, CLL lymphocytes responded to NaIO 4 more extensively and earlier than to PHA.