Ara Tourian

Activation of phenylalanine hydroxylase by phenylalanine.

Abstract 1. 1. Phenylalanine activates phenylalanine hydroxylase ( l -phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.3.1), when dithiothreitol is used to regenerate 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. 2. 2. Phenylalanine hydroxylase activation by phenylalanine follows first order kinetics; the activation rate is time and temperature dependent, and is described by the exponential equation v a = v f + ( v o − v f ) e − at . 3. 3. The kinetic analysis, derived from the effect of phenylalanine on phenylalanine hydroxylase, is consistent with the postulate that the enzyme has a second activating site for phenylalanine distinct from its catalytic site. 4. 4. The sedimentation constant of non-activated phenylalanine hydroxylase is 6.1 and shifts to 8.14 on activation. Activation shifts the equilibrium from a dimer of 110 000 molecular weight to a tetramer of 210 000.

A case of myelinoclastic diffuse sclerosis in an adult.

We report a case of rapidly progressive cerebral demyelinat- ing disease in a previously healthy 40-year-old woman. This case satisfies the diagnostic criteria for myelinoclastic diffuse sclerosis (MDS), but is unusual in the age of onset. This is the 1st case of MDS in an adult with full documentation of clinical, biochemical, radiographic, and pathologic features.

Developmental biology of hepatic phenylalanine hydroxylase activity in foetal and neonatal rats synchronized as to conception

Abstract A biphasic pattern of development of phenylalanine hydroxylase activity is found in foetal and neonatal rats synchronized as to conception. The first burst of activity is at 22 days and 12 h. The second burst activty occurs between 2 and 3 weeks of age. Progesterone decreases the maximal level of enzyme activity achieved during the initial burst. However, the fundamental biphasic pattern seems to be independent of birth, progesterone or maternal milk. Hypophysectomy, thyroidectomy and adrenalectomy do not result in a significant change of enzymatic activity in the 26-day-old rat. The activity of phenylalanine hydroxylase is maintained at a constant steady state during a 24-h cycle in the 6-week-old rat.

Glucosamine dependence of Huntington's chorea fibroblasts in culture

Abstract Huntingtons chorea is an autosomal dominant disease of the nervous system. Fibroblasts of one such case obtained from the Genetic Mutant Repository have a normal growth rate when compared with an age, sex and passage number matched human fibroblast cell line obtained from the same source. However, when the culture medium is depleted of nutrients and non-essential amino-acids added either individually or in combination, the Huntingtons chorea fibroblasts show a dependence for glucosamine in the culture medium for cell survival and replicative capacity. Glutamine cannot be used in place of glucosamine. In fact, there is a further increment of cell morphology and number deterioration by Huntingtons chorea but not normal fibroblasts when glutamine is added to depleted cultures.

Membrane abnormalities of Huntington's Chorea fibroblasts in culture

Abstract Huntingtons Chorea is an autosomal dominant disease of the nervous system. Proliferating fibroblasts of one such case express metabolic and morphological abnormalities in addition to delayed adhesion to plastic substratum when compared to age, sex and passage number matched human fibroblasts when grown in a minimal essential medium supplemented with glycine or serine and the macromolecular fraction of fetal calf serum. The abnormalities expressed by Huntingtons Chorea fibroblasts are fully corrected when the fibroblasts are grown in whole non-filtered fetal calf serum or when 10 −3 M glucosamine is added to the culture medium.

Administration of 5‐hydroxy‐L‐tryptophan to individuals with Huntington's chorea

Huntington’s chorea is a devastating hereditary disorder of the central nervous system characterized by the appearance in adult life of progressive dementia and chorea. The disease is inherited in a single autosomal dominant fashion with complete penetrance.’ The biochemical manifestations of genetic disease may be present at birth or even during in t rau ter ine life. The absence of clinical symptoms at birth and early adult life does not preclude the possibility that adequate pharmacologic challenge will uncover neural networks demonstrating functional impairment. Birkmayer and Hornykiewicz2 gave two patients with Huntington’s chorea 50 mg of D,L-5-hydroxytryptophan, a precursor of serotonin, intravenously, which resulted in an increase in choreiform movements. Lee, Markham and Clark3 extended the previous observation and administered oral 5-hydroxytryptophan in add i t ion t o methysergide (SansertB) and phenelzine (Nardilq and observed a transient and reversible impairment of mental ability, motor function, and abnormal movements. Methysergide was added to block the peripheral effects of serotonin and phenelzine, a monoamine oxidase inhibitor, to block its degradation. These two drugs were added to achieve higher brain serotonin levels. This adverse response to 5-hydroxytryptophan has been interpreted to represent an overresponsiveness of mesencephalic pallidal and limbic systems to serotonin in Huntington’s disease due to release of pallidal activity. This remarkable observation would suggest that progeny of huntingtonians who carry the defective gene, but who are clinically asymptomatic, could similarly show trans i en t and reversible symptoms of chorea, mental impairment, and increased visual-motor reaction time when pharmacologically challenged with 5-hydroxytryptophan. The present study of four documented Huntington’s chorea cases, their two progeny, and a healthy individual was conducted with the double-blind, crossover method to validate the previous observations of Lee, Markham and Clark4 and to extend the pharmacologic provocative test to progeny of huntingtonians. The hope was to develop a method of uncovering persons who carry the defective gene for purposes of genetic counseling and to gain insight into neurotransmitter mechanisms in Huntington’s disease.

Phenylalanine hydroxylase activity in foetal hepatic organ culture

The biphasic pattern of phenylalanine hydroxylase (l-phenylalanine tetrahydropteridine:O2 oxidoreductase (4-hydroxylating) EC 1.14.3.1) activity of foetal and neonatal rat liver is not observed in 20- to 22-day-old hepatic explant culture. Two genetic markers of differentiated hepatic tissue were used to monitor hepatic explant: (a) a high baseline tyrosine aminotransferase activity (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5); and (b) hydrocortisone inducibility of this activity. Pre-existing phenylalanine hydroxylase activity of 22-day-old foetal explants decays with a half-life of 6 h. These observations suggest the need for a hepatic or extrahepatic inductive signal for the synthesis of phenylalanine hydroxylase.

l-Glutamined-Fructose-6-P aminotransferase regulation by glucose-6-P and UDP-N-Acetylglucosamine

Abstractl-Glutamined-Fructose-6-P aminotransferase regulates hexosamine synthesis. An affinity purified human fibroblast aminotransferase and specific radioisotope assays developed by us were used to show an independent inhibition of the aminotransferase by Glucose-6-P. More interestingly, at concentration of UDP-N-Acetylglucosamine and glucose-6-P where either sugar has no independent inhibitory effect, there is an allosteric and significant inhibition of the aminotransferase.

A sensitive, specific radioisotope assay for l-glutamine-d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for L-glutamine-D-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K/sub 1/ concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 ..mu..g of cell-extract protein will yield measurable product. In contrast, 280 ..mu..g of crude-extract protein from colon is required with the modified Elson-Morgan colorimetric assay.

The unique identity of rat hepatoma phenylalanine hydroxylase.

Abstract Rat hepatoma H4-II-E-C3 culture phenylalanine hydroxylase is unique and differs from any of the three isozymes of liver or the one of kidney. Isoelectric focusing results in a single isozyme with an isoelectric point of 5.20 compared to 5.20, 5.30, 5.60 for the liver and 5.35 for the kidney. Hepatoma phenylalanine hydroxylase cross reacts with antibody # 2 of rat liver but differs from isozyme # 2 by a tenfold difference in the ratio of antigen to antibody at equivalence on immunotitration. Kidney phenylalanine hydroxylase which also cross reacts with antibody # 2 of liver can quantitatively (at 2 mU per mg immunoglobulin) absorb antibody against hepatoma enzyme, liver isozyme # 2 and itself without having any effect on antibody # 1 and # 3 of liver.