Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

Codon usage bias analysis of genes linked with esophagus cancer

Esophageal cancer involves multiple genetic alternations. A systematic codon usage bias analysis was completed to investigate the bias among the esophageal cancer responsive genes. GC-rich genes were low (average effective number of codon value was 49.28). CAG and GTA are over-represented and under-represented codons, respectively. Correspondence analysis, neutrality plot, and parity rule 2 plot analysis confirmed the dominance over mutation pressure in modulating the codon usage pattern of genes linked with esophageal cancer.     

p-ethynylphenylalanine: a potent inhibitor of tryptophan hydroxylase

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p-ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N-tertbutyloxycarbonyl-4-iodo-L-phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p-chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 +/- 6.2 microM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate-limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors. Administration of pEPA (30 mg/kg) to rats produced a 95 +/- 5% decrease in TPH activity in brain homogenates and a concomitant decrease in serotonin and 5-hydroxyindole-3-acetic acid levels (85%) at 24 h after injection. In contrast, pCPA produced a similar effect (87 +/- 5% decrease in TPH activity) only at 10 times the concentration (300 mg/kg). These results suggest that pEPA is a selective, reversible, and potent inhibitor of TPH both in vitro and in vivo. The potential for pEPA to inhibit selectively and reversibly the biosynthesis of serotonin may contribute to the characterization of the role of serotonin in behavioral and physiological activities.     

Binding studies on heavy-light chain recombinant hybrid immunoglobulins A derived from murine myeloma anti-fructofuranans.

Heterologous H-L chain recombinants derived from homogeneous murine myeloma immunoglobulins with anti-inulin specificity were found to bind inulin-related oligosaccharides with affinities closely paralleling those of the L chain donors.     

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased V_max with unchanged K_m values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.

     

L-methionine inhibits reaction of DNA with anticancer cis-diamminedichloroplatinum(II)

Sufficient evidence has accumulated to identify DNA as the relevant pharmacological target of antitumor cisplatin [cis-diamminedichloroplatinum(II)]. This drug is administered intravenously so that before it reaches DNA in the nucleus of tumor cells it may interact with various compounds including sulfur-containing molecules such as L-methionine or the compounds containing these residues. L-Methionine increases the rate of reaction of cisplatin with monomeric guanosine 5'-monophosphate, and it was suggested on the basis of these results previously obtained by other authors that methionine residues could mediate the transfer of platinum onto DNA. We studied in the present work the reactions of the 1:1 complex formed between cisplatin and L-methionine or N-acetyl-L-methionine with synthetic, single- and double-stranded oligodeoxyribonucleotides and natural, high molecular mass DNA by using high-pressure liquid chromatography and flameless atomic absorption spectrophotometry. The results demonstrate that both L-methionine and N-acetyl-L-methionine decrease the rate of reaction of cisplatin with base residues in natural, high molecular mass DNA. Thus, the possibility that cisplatin bound to methionine residues serves as a drug reservoir available for platination of DNA in the nucleus of tumor cells appears unlikely.