Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP+) by preparations of pure human liver MAO B:monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the Km,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, Vmax,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce Vmax,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.

Antimalarial activity and inhibition of monoamine oxidases A and B by exo-erythrocytic antimalarials. Optical isomers of primaquine, N-acylated congeners, primaquine metabolites and 5-phenoxy-substituted analogues.

When the terminal amino group in the side chain of primaquine was blocked with an ethoxyacetyl group shown in 2, or eliminated by oxidative deamination to carboxylic acid 3, the antimalarial effect was markedly reduced in a screening assay which measures tissue schizonticidal activity. The optical isomers 1A and 1B of primaquine had similar antimalarial potency to the racemic mixture but 1B appeared less toxic. The 5-phenoxy-substituted analogue 4, belonging to a new class of antimalarials, showed similar potency in the assays to either 1A or 1B but seemed less cytotoxic than (+/-)-primaquine. Compounds 1A and 1B were found to be competitive inhibitors of human monoamine oxidase (MAO) A and B (Ki range 103-225 microM), but 4 showed 10-30-fold greater competitive inhibition of MAO A (Ki = 6.8 microM) and 40-90-fold greater non-competitive inhibition of MAO B (Ki = 2.3 microM).

Inhibition of monoamine oxidases A and B by simple isoquinoline alkaloids: racemic and optically active 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines.

A series of 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines were tested as substrates and/or inactivators of highly purified human monoamine oxidase A and B (MAO A and B). None were found to be a substrate for either enzyme, but many of these isoquinolines could selectively inhibit either MAO A or B. Stereoselective competitive inhibition of MAO A was found with the R enantiomer of all the stereoisomers tested, including salsolinol (Ki = 31 microM), salsoline (Ki = 77 microM), salsolidine (Ki = 6 microM), and carnegine (Ki = 2 microM). As a class, the 3,4-dihydro-isoquinolines were the most potent inhibitors tested (Ki = 2-130 microM), and the fully aromatic isoquinolines had intermediate activity (Ki = 17-130 microM) against MAO A. In contrast, only a few of these compounds markedly inhibited MAO B. 1,2,3,4-Tetrahydroisoquinoline, its 2-methyl derivative, and o-methylcorypalline gave apparent Ki values of 15, 1, and 29 microM, respectively, and two 3,4-dihydroisoquinolines (compounds 22 and 25) showed substantial inhibition of MAO B (Ki = 76 and 15 microM, respectively). These results support the concept that the topography of the inhibitor binding site differs in MAO A and B.

Studies of the in vitro oxidation of 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol and its dehydration product 1,2,3,6-tetrahydro-1-methyl-4-(methylpyrrol-2-yl) pyridine by human monoamine oxidases A and B.

The ability of highly purified preparations of human monoamine oxidase A and B (MAO A and B) to utilize 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol (MMPP) and its dehydration product 1,2,3,6-tetrahydro-1-methyl-4-(methylpyrrol-2-yl) pyridine (TMMP) as substrates was investigated. The results showed that TMMP was a substrate for both forms of MAO with Km,app values of approximately 60 microM. However, MAO B had a Vmax,app for TMMP about 30-fold greater than MAO A. Additional studies revealed that MMPP was a poor substrate of only MAO B (Km,app = 9.5 mM) and that acid treatment of MMPP led to the formation of a product that could be readily oxidized by both MAO A and B. Similar acid pretreatment of TMMP yielded a product that was a much poorer substrate for MAO B than the parent compound. These results may partially explain why orally administered MMPP produces neurotoxicity in monkeys and TMMP fails to induce chemical parkinsonism.

The effect of debrisoquin on MAO A and MAO B activities.

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 microM and liver MAO B with a Ki value of 8.8 microM, 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.

Isolation of two novel adenosine binding proteins from bovine brain.

Adenosine plays many significant roles both as a metabolic precursor and cell communicator. This report describes the preliminary characterization of two adenosine binding proteins isolated from bovine brain membranes. By using N6-9-aminononane adenosine labeled Sepharose 4B two major affinity bound proteins were purified having apparent molecular weights of 16 and 35 kDa. Either or both of the proteins could be selectively eluted from the affinity column with N6-9-aminononane adenosine, adenosine, cAMP, AMP, ADP, ATP, R-/S-phenylisopropyladenosine and NAD(H). By contrast, no proteins were eluted with caffeine, adenine, deoxyadenosine, 2',3'-AMP, inosine, IMP, xanthine, XMP, GMP, GTP or 5'-N-ethylcarboxamideadenosine. The selectivity of elution and lack of apparent enzymatic activity suggests that these proteins are novel membrane bound adenosine binding proteins.

The anabolic effects of insulin on type II collagen synthesis of Swarm rat chondrosarcoma chondrocytes.

The anabolic effects of insulin on collagen production of freshly isolated Swarm rat chondrosarcoma chondrocytes were investigated. The specific radioactivity of newly synthesized collagen was not increased by insulin, indicating that the hormone has no effect on the specific radioactivity of the aminoacyl tRNA pool. Results of further studies obtained from collagen degradation experiments demonstrated that insulin did not alter the rate of [3H]collagen degradation. Together, these results clearly indicate that insulin stimulates collagen biosynthesis. Polyacrylamide gel analysis of the newly synthesized collagen of both control and insulin-stimulated cells revealed a large-molecular-weight component which migrated with authentic alpha 1(II) collagen and was collagenase-sensitive. Additional studies showed that, although insulin increased the processing and secretion of collagen, the hormone did not cause a shift in the distribution of the extracellular and intracellular collagen pools. Finally, results of studies conducted with the transcriptional inhibitor, actinomycin D, indicated that the anabolic effects of insulin on collagen and non-collagen proteins were mediated at a post-transcriptional site.

Synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate in vitro and in vivo.

The pathway of synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate (PP-InsP4-PP) was elucidated by high performance liquid chromatography using newly available 3H- and 32P-labeled substrates. Metabolites were also identified by using two purified phosphatases in a structurally diagnostic manner: tobacco "pyrophosphatase" (Shinshi, H., Miwa, M., Kato, K., Noguchi, M. Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inositol polyphosphate phosphatase (MIPP; Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498). The demonstration that diphosphoinositol polyphosphates were hydrolyzed by MIPP provides new information on its substrate specificity, although MIPP did not metabolize significant amounts of these polyphosphates in either rat liver homogenates or intact AR4-2J cells. In liver homogenates, inositol hexakisphosphate (InsP6) was phosphorylated first to a diphosphoinositol pentakisphosphate (PP-InsP5) and then to PP-InsP4-PP. These kinase reactions were reversed by phosphatases, establishing two coupled substrate cycles. The two dephosphorylations were probably performed by distinct phosphatases that were distinguished by their separate positional specificities, and their different sensitivities to inhibition by F- (IC50 values of 0.03 mM and 1.4 mM against PP-InsP5 and PP-InsP4-PP, respectively). In [3H]inositol-labeled AR4-2J cells, the steady-state levels of PP-[3H]InsP5 and PP-[3H]InsP4-PP were, respectively, 2-3 and 0.6% of the level of [3H]InsP6. The ongoing turnover of these polyphosphates was revealed by treatment of cells with 0.8 mM NaF for 40 min, which reduced levels of [3H]InsP6 by 50%, increased the levels of PP-[3H]InsP5 16-fold, and increased levels of PP-[3H]InsP4-PP 5-fold. A large increase in levels of PP-[3H]InsP5 also occurred in cells treated with 10 mM NaF, but then no significant change to levels of PP-[3H]InsP4-PP were observed; there may be important differences in the control of the turnover of these two compounds.

Identification and purification of diphosphoinositol pentakisphosphate kinase, which synthesizes the inositol pyrophosphate bis(diphospho)inositol tetrakisphosphate.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) were recently identified as inositol phosphates which possess pyrophosphate bonds. The molecular mechanisms that regulate the cellular levels of these compounds are not yet characterized. To pursue this question, we have previously purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants [Voglmaier, S. M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4305-4310]. We now report the identification and purification of another novel kinase, diphosphoinositol pentakisphosphate (PP-IP5) kinase, which uses PP-IP5 as a substrate to form bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) in soluble fractions of rat forebrain. The purified protein, a monomer of 56 kDa, displays high affinity (Km = 0.7 microM) and selectivity for PP-IP5 as a substrate. The purified enzyme also can transfer a phosphate from bis-PP-IP4 to ADP to form ATP. This ATP synthase activity is an indication of the high phosphoryl group transfer potential of bis-PP-IP4 and may represent a physiological role for PP-IP5 and bis-PP-IP4.

The interaction of coatomer with inositol polyphosphates is conserved in Saccharomyces cerevisiae.

Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP6 to mammalian coatomer [Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J. Biol. Chem. 269, 17826-17832] is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 361 +/- 102 nM). Ligand specificity was studied by displacing 4.5 nM [3H]InsP6 from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)-D-Ins(3,4,5)P3 = 290. Less than 10% of the 3H label was displaced by 1 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast [Hawkins, Stephens and Piggott (1993) J. Biol. Chem. 268, 3374-3383], indicate that InsP6 is the major physiologically relevant ligand. Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP6 by coatomer.

Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3.

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer.

The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C. P., Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991) J. Am. Chem. Soc. 113, 3154-3162). Kinetic and physical characterization of the E. coli-expressed protein show that it is identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits.

A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase.

Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.

Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

Biological variability in the structures of diphosphoinositol polyphosphates in Dictyostelium discoideum and mammalian cells.

Previous structural analyses of diphosphoinositol polyphosphates in biological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was determined by NMR to be 4- and/or 6-PPInsP5, and the bisdiphosphoinositol tetrakisphosphate was found to be 4, 5-bisPPInsP4 and/or 5,6-bisPPInsP4 [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315, 715-720]. We now describe three recent technical developments to aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution and sensitivity of detection of PPInsP5 isomers by microbore metal-dye-detection HPLC; (ii) the use of the enantiomerically specific properties of a rat hepatic diphosphatase; (iii) chemical synthesis of enantiomerically pure reference standards of all six possible PPInsP5 isomers. Thus we now demonstrate that the major PPInsP5 isomer in Dictyostelium is 6-PPInsP5. Similar findings obtained using the same synthetic standards have been published [Laussmann, Reddy, Reddy, Falck and Vogel (1997) Biochem. J. 322, 31-33]. In addition, we show that 10-25% of the Dictyostelium PPInsP5 pool is comprised of 5-PPInsP5. The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP5 is the predominant PPInsP5 isomer in four different mammalian cell lines (FTC human thyroid cancer cells, Swiss 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). The fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our technological developments for future determinations of the structures of this class of compounds in other systems.

cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties.

The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, we have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.