Daniel B. Ellis

Biosynthesis of respiratory tract mucins: II. Control of hexosamine metabolism by l-glutamine:d-fructose 6-phosphate aminotransferase☆

1. 1. l-Glutamine:d-fructose 6-phosphate aminotransferase (EC 2.6.1.16) was isolated and partially purified from bovine trachea. The enzyme exhibited a broad pH optima from 6.5 to 7.5. 2. 2. The allosteric inhibition of the enzyme by UDP-N-acetylglucosamine was investigated in detail. A Hill plot of the kinetic data indicated negative cooperativity in the presence of UDP-N-acetylglucosamine. 3. 3. An analogue of l-glutamine, 6-diazo-5-oxo-l-norleucine exhibited competitive inhibition with respect to l-glutamine. 4. 4. A discussion of these findings with reference to the regulation of hexosamine biosynthesis in the mucus-producing structures of the trachea is presented.

Uridine diphosphate N-acetyl-d-glucosamine 2-epimerase from rat liver: I. Catalytic and regulatory properties

Abstract 1. 1. UDP-N-acetylglucosamine 2-epimerase, the enzyme catalyzing the formation of UDP and N-acetylmannosamine from UDP-N-acetylglucosamine, was purified 100–200-fold from rat liver. The final enzyme preparation was extremely unstable with activity completely disappearing within a few hours. 2. 2. The epimerase reaction had double pH optima of 7.1 and 7.9 and a Km of 0.2 mM for UDP-N-acetylglucosamine was found. 3. 3. The allosteric inhibition of the enzyme by CMP-N-acetylneuraminic acid was investigated in detail. A sigmoidal inhibition curve was obtained suggesting cooperative homotropic effects. A Hill plot yielded an interaction coefficient of n = 4 . 4. 4. The regulatory significance of the epimerase reaction to nucleotide sugar metabolism is discussed.

Uridine diphosphate N-acetyl-d-glucosamine 2-epimerase from rat liver: II. Studies on the mechanism of action☆

Abstract 1. 1. Kinetic studies of product inhibition of the reaction catalyzed by UDP- N -acetylglucosamine 2-epimerase were consistent with an ordered mechanism, where UDP was the first product released followed by irreversible formation of N -acetylmannosamine. 2. 2. Double reciprocal plots of initial velocity versus UDP- N -acetylglucosamine concentration at several levels of UDP showed the inhibition produced by UDP was linear noncompetitive with K i slope less than K i intercept . 3. 3. From the evidence presented, a two step mechanism is proposed which involves the elimination of UDP from UDP- N -acetylglucosamine to give a 2-acetamidoglucal intermediate which is subsequently converted to N -acetylmannosamine.

Regulation of L-glutamine:D-fructose-6-phosphate aminotransferase activity in bovine trachea.

Abstract Kinetic studies of l -glutamine: d -fructose-6-phosphate aminotransferase (EC 2. 6.1.16) isolated from bovine trachea show an apparent Km of 1.4 mM for fructose 6-phosphate and 0.21 mM for glutamine. The feedback inhibition pattern by UDP- GlcNAc for this enzyme is competitive with fructose 6-phosphate and noncompetitive with glutamine. Concentations of glutamine above 1.6 mM cause substrate inhibition which is independent of the feedback control.

Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent: 2. In vitro profile

HP 370, 4,9‐dibromo‐6‐(4‐methyl‐1‐piperazinyl)benzo[b]pyrrolo[3,2,1‐jk][1,4] benzodiazepine, a compound that has an atypical antipsychotic profile in vivo, was tested for activity in a number of neurotransmitter receptor binding and uptake assays and compared with serveral typical and atypical antipsychotic compounds. The in vitro profile of HP 370 was most similar to that of clozapine, with the notable exception that HP 370 was inactive as an inhibitor of quinuclidinyl benzilate (QNB) binding (IC50 10−5). HP 370, like clozapine, was a relatively weak and nonselective inhibitor of dopamine (DA) receptor subtypes, whereas sulpiride, another compound reported to be atypical, is a very selective but weak inhibitor of dopamine‐2(D2) receptors. HP 370 and clozapine inhihited WB4101 binding with high affinity, indicating α1‐adrenergic receptor blockade. Neither HP 370 nor the standard neuroleptic compounds inhibited flunitrazepam or gamma‐aminobutyric acid (GABA) binding. HP 370, Clozapine, and sulpiride were inactive as inhibitors of norepinephrine (NE), DA, and 5HT uptake, whereas HP 370 AND clozapine were weark inhibitors of GABA uptake. Based on this in vitro data, the mesolimbic site‐selectivity of HP 370 seen in vivo cannot be explained by anticholinergic GABA‐mimetic activity in the DA‐parative results point to a distinct advantage of an atypical neuroleptic, without the antocholinergic side effects of clozapine.

Biosynthesis of respiratory tract mucins: III. Metabolism of aminosugars by tracheal mucosal extracts☆

Abstract 1. 1. The activities of enzymes involved in the metabolism of aminosugars were measured in a particle-free enzyme preparation obtained from bovine tracheal mucosa. 2. 2. This crude extract was capable of catalyzing the series of reactions involving N -acetylmannosamine and leading to the formation of cytidine monophospho-sialic acid, the immediate precursor of the sialic acid residues of mucin glycoproteins. 3. 3. Uridine diphospho- N -acetylglucosamine 2′-epimerase, the enzyme that catalyzes the de novo synthesis of N -acetylmannosamine, and cytidine monophospho-sialic acid synthetase were further purified for kinetic studies.

The use of high-voltage paper electrophoresis in studies of the biosynthesis of mucin glycoproteins.

Abstract The mammalian respiratory tract produces a secretion in which the major macromolecular components are mucin-type glycoproteins. High-voltage paper electrophoresis has proved a valuable research tool in studies of the formation of these glycoproteins. Separations of hexoses, amino sugars, oligosaccharides, sugar nucleotides and mucin glycoproteins are rapidly and easily obtained by paper electrophoresis at voltages of 50 to 100 V/cm. The use of high-voltage paper electrophoresis to follow amino sugars interconversions and the transfer of glycosyl units to mucin acceptors is described.

Dopaminergic agonist and antagonist effects on striatal tyrosine hydroxylase distribution

Abstract The haloperidol-induced activation of striatal tyrosine hydroxylase (TH) is well established, but the form(s) of the enzyme involved with this response remain(s) obscure. Rats were decapitated one hour after either haloperidol (1 mg/kg) or 30 minutes after apomorphine (2 mg/kg) in the presence or absence of pyrogallol (250 mg/kg) pretreatment. Subcellular fractions were prepared from striatal tissue, and L-DOPA formation was measured using reverse phase, paired-ion HPLC with electrochemical detection. The results indicate that the in vivo administration of haloperidol, but not apomorphine, shifts the subcellular distribution of striatal TH activity. This shift occurred from the cytosol fraction (S2) to the crude mitochondrial fraction (P2). No shift in TH activity was observed within the crude synaptosomal fraction after either drug. It is suggested that the ability of haloperidol to increase dopaminergic activity in vivo is associated with a shift in striatal TH activity distribution.