ABSTRACT
Intraperitoneal injection of [4-36Cl, 2-14C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared a 1--4 moles/mole of [14C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-14C]tyrosine from [2-14C]pCPA without inactivating these enzymes. Injection of L-[(U)-14C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections of p-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase, Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.
Subject(s)
Brain/enzymology , Fenclonine/pharmacology , Liver/enzymology , Phenylalanine Hydroxylase/antagonists & inhibitors , Tryptophan Hydroxylase/antagonists & inhibitors , Animals , Biopterins/analysis , Carbon Radioisotopes , Chlorine , Kinetics , Male , Radioisotopes , Rats , Rats, Inbred StrainsABSTRACT
Repeated intraventricular injections of 2,4-diamino-6-hydroxypyrimidine (DAO-Pyr), inhibitor of D-erythro-q-dihydroneopterin triphosphate synthetase, inhibited q-BH2 synthesis from GTP, markedly increased accumulation of 2-amino-4-hydroxy-5 (or -6)-formamido-6-triphosphoribosylaminopyrimidine (FPyd-P3) and brought about a 60% decrease in the in vivo of reduced biopterin (BH2 and BH4) pool in the brain. Nevertheless, there was no effect on the rate of hydroxylation of L-tryptophan or on the 5-hydroxytryptamine level in rat brain. These data emphasized the significance of the rate of hydrogen transfer and the limitation of the concept of "unsaturation" (i.e., the absolute amount of the carrier pterin molecule) for the synthesis of neurotransmitters in vivo.
Subject(s)
Biopterins/biosynthesis , Biopterins/metabolism , Brain/metabolism , Hypoxanthines/pharmacology , Pteridines/biosynthesis , Pteridines/metabolism , Tryptophan Hydroxylase/metabolism , Animals , Biopterins/analogs & derivatives , Guanosine Triphosphate/metabolism , Pyrimidine Nucleotides/metabolism , Rats , Serotonin/metabolismABSTRACT
One pathway by which tryptophan is metabolized in the brain as well as in the periphery is through cleavage of the indole ring to formylkynurenine and then kynurenine. Indoleamine-2,3-dioxygenase, the enzyme that catalyzes this cleavage, and kynurenine are distributed all across the different anatomic regions of brain. Approximately 40% of the kynurenine in brain is synthesized there, the remainder having come from plasma. Tryptophan loading, which has been used both experimentally and therapeutically as a means of increasing tryptophan conversion to serotonin, also increases kynurenine formation in the brain and in the periphery. Because of the formation of kynurenine, which competes for cerebral transport and cellular uptake of L-tryptophan, and because of substrate inhibition on tryptophan hydroxylase, excessively high doses of tryptophan may actually decrease the production of cerebral serotonin and 5-hydroxyindoleacetic acid.
Subject(s)
Brain/metabolism , Kynurenine/metabolism , Animals , Arylformamidase/metabolism , Brain/enzymology , Decarboxylation , Humans , Hydroxyindoleacetic Acid/metabolism , Kinetics , Kynurenine/biosynthesis , Kynurenine/blood , Kynurenine/pharmacology , Kynurenine 3-Monooxygenase , Mixed Function Oxygenases/metabolism , Oxygenases/metabolism , Serotonin/metabolism , Tryptophan/blood , Tryptophan/metabolism , Tryptophan/pharmacology , Tryptophan OxygenaseSubject(s)
Brain/metabolism , Mitochondria/metabolism , Norepinephrine/metabolism , Synaptosomes/metabolism , Tryptophan/metabolism , Adenosine Diphosphate , Animals , Centrifugation, Density Gradient , Electron Transport Complex IV/metabolism , Ficoll , Fumarate Hydratase/metabolism , In Vitro Techniques , Kinetics , L-Lactate Dehydrogenase/metabolism , Malates , Male , Oligomycins/pharmacology , Oxygen Consumption , Pyruvates , RatsABSTRACT
An enzyme that catalyzes the conversion of 2-amino-6-(5'-triphosphoribosyl)amino-5- or 6-formamido-6-hydroxypyrimidine, but not of guanosine triphosphate, to quinonoid 6-(D-erythro-1'-2'-3'-trihydroxypropyl)dihydropterin triphosphate and formic acid has been purified to homogeneity from some mammalian brain and liver. The enzyme of a single strand is a basic protein of 9177 daltons consisting of 68 amino acid residues--except the enzyme from rat brain, which has one additional aspartic acid as residue 7. The enzyme possesses three free SH groups and, in its most active form, 1 mol of phosphate per mole of enzyme. Peptides isolated after hydrolysis with trypsin, chymotrypsin, or weak acid were separated by thin-layer chromatography and sequenced manually by Edman degradation. The complete sequence of the molecule was established as follows: (formula: see text)
Subject(s)
Biopterins/biosynthesis , Pteridines/biosynthesis , Amino Acid Sequence , Animals , Biopterins/metabolism , Brain/enzymology , Cattle , Chickens , Guinea Pigs , Molecular Weight , Rabbits , Rats , Sulfhydryl Compounds/analysisSubject(s)
Alcohol Oxidoreductases/deficiency , Phenylketonurias/etiology , Animals , Biopterins/analogs & derivatives , Brain/enzymology , Cattle , Guinea Pigs , Humans , Rabbits , RatsSubject(s)
Biopterins/biosynthesis , Brain/metabolism , Pteridines/biosynthesis , Animals , Biopterins/analogs & derivatives , Cytosol/metabolism , Guanosine Triphosphate/metabolism , Humans , Microsomes/metabolism , Mitochondria/metabolism , Neurons/metabolism , Organ Specificity , Rabbits , Rats , Species Specificity , Subcellular Fractions/metabolism , Synaptosomes/metabolismSubject(s)
Biopterins/biosynthesis , Brain/enzymology , Liver/enzymology , Pteridines/biosynthesis , Animals , Biopterins/analogs & derivatives , Cattle , Kinetics , Phosphorylation , RatsABSTRACT
Three specific enzymes are involved in the cerebral synthesis of 7,8-dihydrobiopterin from GTP. These were isolated, purified, and characterized. The first enzyme, also catalyzing the rate-limiting step, is GTP-cyclohydrolase A-I or Mg2+-dependent A-II, which hydrolyze the GTP to the specific product 2-amino-6-(5-triphosphoribosyl)-amino-5-or-6-formamido-6-hydroxypyrimidine (FPyd-P3). FPyd-P3 is cyclized by a synthetase to D-erythro-7,8-dihydroneopterintriphosphate (NPTH2-P3). The new enzyme, D-erythro-7,8-dihydroneopterintriphosphate synthetase (enzyme B) is a basic protein of 9177 daltons containing three free SH groups, isoleucyl-seryl- as N- and valyl-glutamyl- as C-terminals. This enzyme of 69 amino acid residues from rat and 68 residues (one less aspartic acid) from guinea pig brain contains no hydroxyproline, methionine, or tryptophan. The enzyme from rat brain will gradually convert its product NPTH2-P3 to BH2, wherease the enzyme from guinea pig brain lacks this property. 2,4-amino-6-hydroxypyrimidine and dFPyd-P3 are effective inhibitors of this enzyme. The synthesis of BH2 from NPTH2-P3, but not from 7,8-dihydroneopterin, is catalyzed by L-erythro-7,8-dihydrobiopterin synthetase (enzyme C), which was purified to electrophoretic purity. This enzyme does not require pyridine nucleotides of Mc2+ for its catalysis.