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Monoamine oxidase A and B (MAO A and B) are the major neurotransmitter-degrading enzymes in the central nervous system and in peripheral tissues. MAO A and B cDNAs from human, rat, and bovine species have been cloned and their deduced amino acid sequences compared. Comparison of A and B forms of the enzyme shows approximately 70% sequence identity, whereas comparison of the A or B forms across species reveals a higher sequence identity of 87%. Within these sequences, several functional regions have been identified that contain crucial amino acid residues participating in flavin adenine dinucleotide (FAD) or substrate binding. These include a dinucleotide-binding site, a second FAD-binding site, a fingerprint site, the FAD covalent-binding site, an active site, and the membrane-anchoring site. The specific residues that play a role in FAD or substrate binding were identified by comparing sequences in wild-type and variants of MAO with those in soluble flavoproteins of known structures. The genes that encode MAO A and B are closely aligned on the X chromosome (Xp11.23), and have identical exon-intron organization. Immunocytochemical localization studies of MAO A and B in primate brain showed distribution in distinct neurons with diverse physiological functions. A defective MAO A gene has been reported to associate with abnormal aggressive behavior. A deleterious role played by MAO B is the activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a proneurotoxin that can cause a parkinsonian syndrome in mammals. Deprenyl, an inhibitor of MAO B, has been used for the treatment of early-stage Parkinson's disease and provides protection of neurons from age-related decay.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

Monoamine oxidases A and B (MAO-A and B) catalyze the oxidative catabolism of biogenic amines and xenobiotics. Investigation of these mitochondrial membrane proteins shows that they differ in substrate preference, inhibitor specificity, tissue and neuronal cell distribution, immunological properties, and nucleotide and deduced amino acid sequences. Comparisons of MAO-A and B from the human, bovine, and rat species show strikingly high similarity (85-88%) in the amino acid sequences of each enzyme. Furthermore, three regions in MAO-A and B have sequence identities across species of 78, 88, and 86%. These regions correspond to a nucleotide-binding site near the N-terminal end that is found in the vast majority of enzymes that require flavin adenine dinucleotide (FAD), a region of unknown function, and the FAD-binding site toward the C-terminal end. Genomic clones of MAO-B which span almost the entire gene (greater than 40 kb) have been isolated, restriction mapped, and partially sequenced. Likewise, genomic clones of MAO-A that correspond to the 3'-flanking region have also been investigated. Current studies which focus on identification of the promoter and regulatory sequences should help to establish why MAO-A and B are localized in different subsets of neurons in brain.

A serum factor (SF), isolated from acidified sera of normal donors, inhibited phytohemagglutinin (PHA) stimulation of lymphocytes isolated from patients with chronic lymphocytic leukemia (CLL),but had no effect on those from healthy donors. An SF isolated by an identical procedure from the sera of patients with CLL had essentially no inhibitory effect on DNA synthesis in normal or leukemic lymphocytes. The SF was isolated from sera or plasma by ammonium sulfate precipitation, acidification to pH 1.5, and chromatography on DEAE-cellulose. The SF was heat labile and sensitive to digestion by trypsin. Maximum inhibition was obtained when the factor was added within 24 hours after the cells were stimulated with PHA.

Antibodies in both their free and immobilized state have been the object of considerable industrial and academic interest. A variety of methods are used for preparing and immobilizing antibodies. Applications for monoclonal antibodies include the preparation of therapeutics, diagnostics, and in affinity fractionation. Recent US patents on monoclonal and immobilized antibodies and scientific literature on monoclonal antibodies are surveyed. A description of these patents and a list of references are given.

The effects of fluctuations of free amino acid concentrations in plasma on sleep patterns and operant behavior in the squirrel monkey were studied. Plasma phenylalanine (PHE) and tyrosine (TYR) were rapidly lowered to trace levels within 4 hr by intraperitoneal administration of phenylalanine ammonia-lyase (PAL), an enzyme which specifically deaminates both PHE and TYR to inactivate products. Significant alterations in sleep patterns and in performance on a chained operant task involving hold and reaction time components were found, but no significant effect on the performance of a simple operant task was observed. Adminstration of saline or trans-p-cinnamic acid and trans-p-coumaric acid, the products of PHE and TYR deamination, produced no changes in behavior or sleep patterns. The reduction of plasma PHE and TYR resulted in a significant decrease in PHE and TYR levels in whole rat brain. Brain serotonin levels were increased within 4 hr after PAL administration, whereas, dopamine and norepinephrine levels were decreased subsequently (within 8 hr). These studies suggest that circulating levels of PHE and TYR are involved directly or indirectly in the modulation of certain parameters of brain function.

Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.

Dihydropteridine reductase from human liver and rat striatal synaptosomes is noncompetitively inhibited by apomorphine and its analogs. The Ki or I50 values are in the range of 0.6 to 2.9 microM for R-(-)-apomorphine, R-(-)-and S-(+)-2, 10, 11-trihydroxyaporphine, R-(-)-norapomorphine, R-(-)-N-hydroxyethylnorapomorphine, R-(-)-2,10,11-trihydroxy-N-n-propylnoraporphine, R-(-)- and S-(+)-N-n-propylnorapomorphine, and R-(-)-N-chloroethylnorapomorphine; and 13 to 151 microM for R-(-)-2,11-dihydroxy- 10-methoxyaporphine, R-(-)-apocodeine, and S-(+)-bulbocapnine. Structure-activity studies reveal that 10,11-dihydroxy substitution of the D ring of apomorphine is required for the inhibitory effectiveness of these aporphines. Methylation of the 10-hydroxy group reduces, whereas the 2-hydroxyl substitution of the A ring enhances, their inhibitory potency. N-Alkylation variably affects the inhibitory potency of aporphines. In addition, S-(+)-enantiomers of aporphines and dopaminergic antagonists are equally potent as inhibitors of this enzyme, as compared to the corresponding R-(-)-enantiomers and other aporphine agonists. Haloperidol (0.1 to 10 microM) failed to reverse the enzyme inhibitory effectiveness of apomorphine when it was incubated with intact rat striatal synaptosomes prior to or after the addition of apomorphine (0.5 to 1 microM). These results suggest that the inhibitory effects of apomorphine and its analogs against this enzyme are not mediated by their stimulation of dopamine autoreceptors. Since dihydropteridine reductase is required in vivo for the hydroxylation of tyrosine, the inhibition of this enzyme by apomorphine may represent one of several mechanisms by which apomorphine inhibits catecholamine synthesis.

PERIPHERAL blood lymphocytes in culture undergo cell division in the presence of the mitogenic stimulant phyto-haemagglutinin (PHA)1. Some of the metabolic processes that occur shortly after stimulation include phosphorylation of membrane bound phosphatidyl inositol2, acetylation of histones3, changes in cyclic GMP levels4, a pronounced elevation of uridine kinase activity5 and increases in RNA synthesis6 and protein synthesis7. These events are followed by the initiation of DNA replication within approximately 24 h8,9. Frequently, labelled uridine and thymidine are used as nucleic acid precursors to measure RNA and DNA biosynthesis. In one of these studies, Lucas observed that additions of cytidine inhibited the incorporation of labelled uridine into RNA in PHA-stimulated lymphocytes5. This finding was confirmed in this laboratory and these studies were expanded to include the effects of other nucleosides such as guanosine on lymphocytes from healthy donors and from patients with chronic lymphocytic leukaemia (CLL)10.

A serum factor (SF), isolated from acidified sera of normal donors, inhibited phytohemagglutinin (PHA) stimulation of lymphocytes isolated from patients with chronic lymphocytic leukemia (CLL),but had no effect on those from healthy donors. An SF isolated by an identical procedure from the sera of patients with CLL had essentially no inhibitory effect on DNA synthesis in normal or leukemic lymphocytes. The SF was isolated from sera or plasma by ammonium sulfate precipitation, acidification to pH 1.5, and chromatography on DEAE-cellulose. The SF was heat labile and sensitive to digestion by trypsin. Maximum inhibition was obtained when the factor was added within 24 hours after the cells were stimulated with PHA.

Lymphocytoid and plasmacytoid blasts have been identified in both normal and chronic lymphocytic leukemia lymphocyte cultures exposed to NaIO4. However, the appearance of ultrastructurally abnormal blasts in chronic lymphocytic leukemia cultures suggests that NaIO4 also stimulates the transformation of an abnormal subpopulation of lymphocytes. Development of invaginated nuclei produced morphological features similar to nuclear blebs, a cellular abnormally described in blood cells from other cancers. Furthermore, the consistent localization of nuclear invagination only to portions of nuclei adjacent to developing cytoplasmic microtublar complexes suggests a role for microtubules in the transformation process. The absence of these unusual blasts in cultures stimulated with other mitogens may indicate that these NaIO4-sensitive cells are not responsive to the more commonly used plant lectins.

Monoamine oxidase B (MAO B; EC 1.4.3.4) activity in detergent extracts of mitochondria from autopsy brain (gray matter and medulla), liver, lung, and kidney from a single individual and from pooled, human platelets could be immunoprecipitated by a monoclonal anti-human platelet MAO B antibody (MAO-1C2) in combination with appropriate secondary reagents. MAO A activity, which was detected in brain, liver, lung, and kidney, was not immunoprecipitated under the same conditions. All MAO B-containing extracts, regardless of tissue source, inhibited immunoprecipitation of [3H]pargyline-labeled human platelet MAO, and the shapes of the inhibition curves were identical. The concentration of immunologically detectable MAO B protein in the extracts was estimated from immunoprecipitation competition data by reference to a standard curve relating observed inhibition of immunoprecipitation to the concentration of catalytically active platelet MAO added (estimated from [3H]pargyline binding data). MAO B protein concentrations measured by this radioimmunoassay were similar to concentrations of active MAO B as measured by pargyline binding. These results demonstrate that in the brain and peripheral tissues studied, molecules with MAO B activity share a unique antigenic determinant and similar catalytic efficiency. They also extend previous observations that MAO B molecules extracted from mitochondria bear an antigenic determinant which is not present on MAO A molecules. These results demonstrate the validity of a new competitive radioimmunoassay for active plus inactive MAO B concentration in human platelet extracts and extracts of mitochondria from human tissues. This radioimmunoassay should complement [3H]pargyline binding assays and enzyme activity assays in studies designed to clarify the mechanisms of genetic, disease, and treatment factors which lead to differences in MAO B function among individuals.

The capacity of skin fibroblasts from cystic fibrosis (CF) and non-CF individuals to incorporate exogenous L-fucose into various metabolic pools was compared. Incorporation into intracellular acid-soluble pools and extracellular glycopeptides was measured by a dual-label co-chromatographic technique, whereas total macromolecular incorporation was measured by a single-label procedure. Although CF and normal fibroblasts incorporate exogenous fucose into intracellular acid-soluble and high molecular weight compounds in a similar manner, CF cells appear to be characterized by an increased incorporation of label into low molecular weight extracellular glycopeptides.

There were no significant differences noted between bipolar manic-depressive patients and normal controls for plasma phenylalanine or tyrosine following an L-phenylalanine loading test given to determine if some affective illness may be related to the heterozygous phenotypic expression of phenylketonuria (reduced liver phenylalanine hydroxylase). The test was able to distinguish known PKU heterozygotes from the other subjects. It is possible that other heterozygous states may be implicated in the development of some psychiatric disorders.

L-Phenylalanine ammonia-lyase (PAL) from yeast was used to deplete plasma L-phenylalanine and L-tyrosine in an attempt to achieve inhibition of tumor growth in mice. Plasma L-phenylalanine and L-tyrosine were reduced to nondetectable levels when circulating PAL activity was maintained at greater than or equal to 0.06 unit/ml. Repeated administration resulted in the appearance of anti-PAL antibodies. A radioimmunoassay based on the method of Farr was developed to determine quantitatively the presence of anti-PAL. Sublethal total-body irradiation temporarily suppressed the immunologic response of the host. Long-term specific immunosuppression to PAL was achieved with cyclophosphamide (CPA). A single dose of 180 mg/kg of CPA administered ip to mice 24 hours before, simultaneously with, or 24 hours after 100 units/kg of PAL induced tolerance for 450 days (20 injections of enzyme). The plasma half-life of PAL in CPA-treated mice remained essentially the same as that found after a single injection (25 hours), and anti-PAL probably will require specific immunosuppression of the host to repeated injections of the enzyme.

Yeast phenylalanine ammonia-lyase was administered i.p. to normal and tumor-bearing mice, and its clearance from plasma was studied. Single and multiple weekly injections at dosages of 10,20,50 and 100 units/kg were administered to C57BL female, C57BL X DBA/2F1 male, and A/J female mice. L5178Y murine lymphoblastic leukemia, B16 melanoma, BW10232 adenocarcinoma, and 15091A anaplastic carcinoma were implanted 7 to 11 days prior to enzyme injection in the appropriate host. After a single injection, the average plasma half-lives of phenylalanine ammonia-lyase were 18 to 24 hr in all groups studied. While the other tumors had no effect on the plasma level of phenylalanine ammonia-lyase after a single injection, L5178Y murine lymphoblastic leukemia and 15091A anaplastic carcinoma significantly depressed the maximal level of phenylalanine ammonia-lyase attained in the plasma. After repeated injections of phenylalanine ammonia-lyase, the initial plasma enzyme level was significantly reduced when 20 units/kg were administered, and the clearance of the enzyme from the plasma was greatly accelerated regardless of the amount administered. Furthermore, in tumor-bearing mice, the rate of clearance was significantly more rapid than in the appropriate non-tumor-bearing control.

We have isolated a mouse monoclonal antibody to human platelet monoamine oxidase (MAO) B. The antibody (MAO-1C2) was isolated from a fusion of mouse myeloma P3/X63 Ag8 to spleen cells from a BALB/c mouse immunized with a partially purified platelet preparation in which an estimated 21-31% of the protein was [3H]pargyline-labeled MAO B. The antibody indirectly immunoprecipitates both [3H]pargyline-labeled, catalytically inactive human MAO B, and unlabeled, catalytically active human MAO B. Binding of the antibody to MAO B has no detectable effect on catalytic activity. MAO-1C2 is specific for human MAO B, and fails to immunoprecipitate MAO A indirectly from human placenta or liver. Its ability to immunoprecipitate human MAO B but not MAO A from extracts of human liver provides a convenient technique for separating the two forms of the enzyme for comparative studies. The antibody does not recognize mouse liver MAO B, suggesting that the determinant is not universally expressed on MAO B from all species.

Tyrosine hydroxylase (TH, tyrosine 3-monooxygenase; EC 1.14.16.2) activity in crude extracts of rat pheochromocytoma, rat brain, and bovine adrenal medulla can be immunoprecipitated in an indirect assay by monoclonal antibodies prepared against partially purified rat pheochromocytoma TH. One of tese monoclonal antibodies, TH-2D8hyphen;2, can be used for immunocytochemical localization of TH in cell bodies, dendrites, and axons in catecholaminergic neurons (e.g., cells in the substantia nigra) of rat brain and in the cell body, neurites, and growth cones of rat pheochromocytoma cells after treatment with nerve growth factor. When linked to Affihyphen;gel 10, this monoclonal antibody can also be used for immunoaffinity purification of rat and bovine TH. These results suggest that THhyphen;2D8hyphen;2 is a valuable reagent with which to investigate the localization, physiological regulation, and function of this important enzyme.

The role of cyclic adenosine 3':5'-monophosphate (cyclic 3':5'-AMP) in the regulation of cell division in lymphocytes obtained from healthy donors and from patients with chronic lymphocytic leukemia (CLL) was investigated by determining the levels of cyclic 3':5'-AMP and glycogen and also the activities of several enzymes that are closely associated with the metabolism of these cellular components. Intracellular levels of cyclic 3':5'-AMP were measured in normal and CLL lymphocytes in nondividing, dividing, and quiescent [after phytohemagglutinin (PHA) addition]states. In normal lymphocytes the levels of cyclic 3':5'-AMP fluctuated throughout the cell cycle after PHA addition, whereas in CLL lymphocytes the levels were approximately 3-fold lower than in normal cells and remained relatively constant before, during, and after mitogenic stimulation. Normal cells contained approximately 3-fold lower levels of glycogen than CLL cells, whereas glycogen phosphorylase activities were increased 2- to 4-fold above those in nondividing cells in normal but not in CLL lymphocytes after stimulation with PHA. Furthermore, cyclic 3':5'-AMP phosphodiesterase activities were higher in CLL lymphocytes than in normal ones. Collectively, these studies demonstrated that (a) the intracellular levels of cyclic 3':5'-AMP differ in these two cell types; (b) the levels of cyclic 3':5'-AMP and glycogen qualitatively correlate with the activities of the enzymes that are related to these components; and (c) an inverse relationship between the levels of cyclic 3':5'-AMP and cell growth exists in mitogen-stimulated lymphocytes from healthy donors but not from patients with CLL. These biochemical differences are presumed to exist between normal and "leukemic" lymphocytes, but alternatively they may reflect normal populations of immunologically distinct lymphocytes.

Monoamine oxidase content in extracts of human blood platelets was determined independently of MAO activity measurements with a recently developed monoclonal antibody against human platelet monoamine oxidase (MAO B) in a competitive radioimmunoassay. Administration of a single oral dose of the irreversible inhibitor tranylcypromine to normal human subjects resulted in a rapid and marked inhibition of platelet MAO catalytic activity within 1 day, followed by recovery of activity to normal levels within 2 weeks. In contrast, there was no change in the net concentration of MAO protein during this time. Since recovery of MAO B activity approximated the reported half-life of blood platelets, these results suggest that recovery of platelet MAO activity after tranylcypromine treatment is due to the replacement of old platelets by new ones which contain catalytically active MAO B.

This chapter describes certain steps in the synthesis and degradation of dopamine and how alterations in them may lead to changes in neuro- transmission and behaviour. The ideas discussed here reflect the collective efforts of many of my colleagues at The University of Texas Medical Branch at Galveston, who have contributed significantly to an integrated programme of biochemical, pharmacological, psychiatric, and behavioural studies of schizophrenic patients, paralleled by correlative studies in animal models. My discussion will include well established principles in the process of neurotransmission, and some innovative hypotheses which are currently being tested.

To examine the mode of action of debrisoquin (DEB), we studied the effect of this drug in vitro on MAO A and MAO B enzyme activities. DEB was shown to be a competitive inhibitor of highly purified human MAO A and MAO B enzyme activities. DEB inhibited placental MAO A with a Ki value of 0.5 μM and liver MAO B with a Ki value of 8.8 μM, 18-fold greater effect on the A form. Kynuramine was used as substrate for both enzymes. Additional studies using a dilution technique showed that DEB was a reversible inhibitor of both forms of the enzyme. The results of this study show that DEB is a potent competitive and reversible inhibitor of both MAO A and MAO B enzymes.

Dihydropteridine reductase from human liver and rat striatal synaptosomes is noncompetitively inhibited by apomorphine and its analogs. The Ki or I50 values are in the range of 0.6 to 2.9 microM for R-(-)-apomorphine, R-(-)-and S-(+)-2, 10, 11-trihydroxyaporphine, R-(-)-norapomorphine, R-(-)-N-hydroxyethylnorapomorphine, R-(-)-2,10,11-trihydroxy-N-n-propylnoraporphine, R-(-)- and S-(+)-N-n-propylnorapomorphine, and R-(-)-N-chloroethylnorapomorphine; and 13 to 151 microM for R-(-)-2,11-dihydroxy- 10-methoxyaporphine, R-(-)-apocodeine, and S-(+)-bulbocapnine. Structure-activity studies reveal that 10,11-dihydroxy substitution of the D ring of apomorphine is required for the inhibitory effectiveness of these aporphines. Methylation of the 10-hydroxy group reduces, whereas the 2-hydroxyl substitution of the A ring enhances, their inhibitory potency. N-Alkylation variably affects the inhibitory potency of aporphines. In addition, S-(+)-enantiomers of aporphines and dopaminergic antagonists are equally potent as inhibitors of this enzyme, as compared to the corresponding R-(-)-enantiomers and other aporphine agonists. Haloperidol (0.1 to 10 microM) failed to reverse the enzyme inhibitory effectiveness of apomorphine when it was incubated with intact rat striatal synaptosomes prior to or after the addition of apomorphine (0.5 to 1 microM). These results suggest that the inhibitory effects of apomorphine and its analogs against this enzyme are not mediated by their stimulation of dopamine autoreceptors. Since dihydropteridine reductase is required in vivo for the hydroxylation of tyrosine, the inhibition of this enzyme by apomorphine may represent one of several mechanisms by which apomorphine inhibits catecholamine synthesis.

Although similar fractions of cells were in the S phase of the cell cycle, normal human skin fibroblasts were shown to incorporate more than twice the 3HTdR into their DNA in vitro than did cells obtained from individuals with cystic fibrosis (CF). Obligate heterozygotes incorporated an intermediate amount of the DNA precursor. Studied were initiated to determine the basis of the differential incorporation of 3HTdR among the genotypes. An analog of thymidine, BUdR, produced varied effects on the growth kinetics of the three genotypes. The growth of cells in BUdR resulted in a 50% increase in the population doubling times of all three genotypes, and caused the cell morphology to change from a spindle shape to one in which the cells became broadened and flat, with numerous cytoplasmic projections extending for distances of several cell diameters. The activities of thymidine kinase and the participation of the exogenous and de novo pathways in the synthesis of TMP were found to be approximately the same in all three genotypes. The data suggest that an alteration in the transport of thymidine into the cells may account for the differences in TdR incorporation into DNA, and this may be associated with other changes in cystic fibrosis that are apparently membrane associated.

Dopamine-derived tetrahydroisoquinolines, such as 3',4'-deoxynorlaudanosolinecarboxylic acid, higenamine-1-carboxylic acid, higenamine, and salsolinol, inhibit human liver dihydropteridine reductase noncompetitively with Ki values ranging from 1.5 to 90 microM. The enzyme is also inhibited noncompetitively by dopamine (Ki = 6 microM) and aminopterin (Ki = 100 microM) but uncompetitively by phenylpyruvic acid (Ki = 6.5 mM). These alkaloids may alter monoamine metabolism in mammals by inhibiting dihydropteridine reductase.

Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.