Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4. Maximum activity of the enzyme was observed at pH 7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.     

Mechanism of the enzymatic reaction catalyzed by uridine diphosphate glucose dehydrogenase. The chemical synthesis of uridine diphosphate glucose-6-H3 and its oxidation by uridine diphosphate glucose dehydrogenase]

The synthesis of UDP-glucose-6-s-H was performed through condensation of alpha-D-glucopyranosyl phosphate-6-3-H and uridine 5'-phosphomorpholidate. Enzymic oxidation of UDP-glucose-6-3-H with calf liver UDP-glucose dehydrogenase was found to proceed with direct transfer of the hydrogen from C-6 of UDP-glucose onto NAD.     

Human plasma dopamine beta-hydroxylase. Purification and properties

Dopamine beta-hydroxylase was isolated from normal human plasma. The major form of the active enxyme in plasma was purified to apparent homogeneity and is a 300,000-dalton tetramer containing 4 atoms of tightly bound copper. About 20% of the enzyme activity in plasma was isolated as a dimeric form of this enzyme. Sodium dodecyl sulfate gel electrophoresis of the purified form gave a polypeptide subunit molecular weight of 72,000 and disulfide-linked dimers of this component were observed. Both forms of the enzyme are apparently glycoproteins and interact with immobilized concanavalin A. Furthermore, the enzyme is capable of binding to alkyl-substituted agarose by hydrophobic interaction. Advantage was taken of these properties to purify the enzyme. Both purified tetramer and partially purified dimer were further characterized by kinetic analysis and the Stokes radii and S20,W of these species were compared. Rabbit antiserum to the purified tetramer revealed no immunochemical differences between the two enzyme forms by using a method of immunotitration.     

Human plasma platelet-activating factor acetylhydrolase. Purification and properties

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.     

Structural requirements in the reaction of morphine uridine diphosphate glucuronyltransferase with opioid substances.

The influence of the 3-hydroxyl and N-alkyl groups in the reactivity of narcotic compounds with morphine UDP-glucuronyltransferase was studied. Opioids possessing both, one or none of these groups were tested for inhibition of morphine glucuronidation in rabbit liver microsomal preparations. Compounds with only a 3-hydroxyl group (normorphine) or an N-methyl group (codeine, ethylmorphine) were less potent competitive inhibitors than those containing both groups (dextrorphan). Norcodeine, with neither of these groups, had no inhibitory effect. The synthetic narcotics (+)- and (-)-methadone, (-)-alpha-acetylmethadol and meperidine, with only an N-alkyl group, were effective competitive inhibitors. No stereoselectivity of the morphine glucuronyltransferase for opioid isomers was observed, and [methionine]enkephalin does not react with morphine glucuronyltransferase. Differences of pKa values and water/lipid solubility of narcotics could not explain the effects. Results indicate that the N-alkyl group plays a critical role in the interaction of narcotics with the morphine UDP-glucuronyltransferase.     

Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli.

The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase of Escherichia coli was over-produced in strains that harbour recombinant plasmids bearing the murD gene under the control of the lac or PR promoter. Purification to homogeneity was achieved by a two-step procedure from a 181-fold over-producing strain. The N-terminal sequence of the purified protein was determined and correlated with the nucleotide sequence of the murD gene. The purified activity was highly dependent on the concentration of potassium phosphate and Mg2+. The enzyme also catalysed the reverse reaction. The Km values for UDP-N-acetylmuramoyl-L-alanine; D-glutamate and ATP/Mg2+ were estimated at 7.5, 55 and 138 microM, respectively. Under the most optimal in vitro conditions determined, a turnover number of 931 min-1 was estimated. When considering the plasmid-free parental strain, the copy number of the murD gene product was not more than 1000.cell-1.     

Enzymatic formation of uridine diphosphate N-acetyl-D-mannosamine.

An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. The optimum pH was between 7.5 and 8.0. At equilibrium of the reaction, the ratio of UDP-N-acetylglucosamine to UDP-N-acetylmannosmaine was about 9:1. The enzyme was inactive toward free N-acetylhexosamines, their phosphate esters, UDP-glucose, and UDP-N-acetylgalactosamine. A stimulatory role of UDP-N-acetylglucosamine was demonstrated. In the reaction with UDP-N-acetylglucosamine, the rate as a function of substrate concentration showed a sigmoidal relationship with a Hill coefficient of 1.8 and an apparent Km value for UDP-N-acetylglucosamine of 1.1 mM. The reverse reaction with UDP-N-acetylmannosamine required the presence of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine concentration required for half-maximal activation was about 0.5 mM. The apparent Km for UDP-N-acetylmannosamine measured in the presence of 0.5 mM UDP-N-acetylglucosamine was 0.22mM. Other nucleotides or hexosamine derivatives were not stimulatory. The same activity was found in cell extracts from several bacterial species.     

Human monocyte carboxylesterase. Purification and kinetics

Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation. Membrane-localized carboxylesterase (CBE) was extracted with nonionic detergent (Triton X-100) and purified by ion exchange (DEAE-cellulose), gel filtration (Sephacryl S-300), hydroxylapatite column, and high performance liquid chromatography. The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretic patterns of the enzyme on polyacrylamide gels run a neutral pH did not vary during enzyme purification. At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8. Pure human monocyte CBE hydrolyzed short chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Purification and properties.

Fructose diphosphate aldolase has been purified to homogeneity from Mycobacterium smegmatis. Physicochemical studies showed that the enzyme is a tetramer of molecular weight 158,000. Mycobacterium smegmatis aldolase, though a bacterial enzyme, possesses properties similar to other class I aldolases. Inactivation of the enzyme by sodium borohydride in presence of dihydroxyacetone phosphate suggested the formation of a Schiff-base intermediate.