David Sidney Feingold

Biosynthesis of heparin

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3′-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.

Isolation of a Golgi-rich fraction from rat liver

Abstract A fractiion rich is membranes of the Golgi apparatus was isolated from rat liver by discontinuous density gradient centrifugation. Electron microscopic analysis of the fraction revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements. The Golgi-rich fraction contained over 90% of the UDP-galactose; N- acetylglucosamine galactosyltransferase, about 2% of the glucose-6-phosphatase and 12% of the AMP phosphohydrolase present in the post-nuclear supernatant of liver homogenates.

Purification and properties of UDPG dehydrogenase from beef liver

UDPG dehydrogenase (UDPglucose:NAD oxidoreductase, EC 1.1.1.22) has been purified from fresh beef liver homogenate by extraction at pH 4.9, ammonium sulfate fractionation between 0.3–0.5 saturation, treatment at 60 °, and pH 4.9 for 1 min, refractionation with ammonium sulfate between 0.35–0.45 saturation, adsorption onto and fractional elution from calcium phosphate gel, chromatography on carboxymethyl cellulose, and finally, chromatography on Sephadex G-200. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and agar immunodiffusion. Upon sedimentation in a sucrose density gradient there was complete coincidence between protein and enzyme activity. Ultracentrifugal sedimentation velocity analysis at pH 7.0 showed a symmetrical peak with a S20,w of 12.8; at pH 5.5 two peaks corresponding to 13 and 20.4 S20,w were present. The turnover number of the enzyme, assuming a molecular weight of 3 × 105, under optimal conditions at 30 ° was 1050 moles UDP-glucose oxidized per minute per mole enzyme. Dehydrogenase activity against dTDP-glucose and GDP-glucose is due to the same protein as that catalyzing the oxidation of UDP-glucose. No activity could be detected with UDP-galactose, UDP-mannose, or GDP-mannose.

Conformational aspects of the reaction mechanisms of polysaccharide lyases and epimerases.

During the biosynthesis of some polysaccharides (e.g. alginic acid [l] and heparin [2]) pyranosyluronate residues undergo epimerization at C-5 after incorporation into the polymer chain. In the case of heparin, the enzyme heparosan-llrsulfate D-glucuronosyl 5-epimerase (EC 5.1.3.17) catalyzes the exchange of hydrogen atoms at C-5 with solvent protons. Hence, it was postulated that the initial step of the epimerization required abstraction of the C-5 proton by a nucleophilic group of the enzyme to form a carbanion; the carbanion could regain a proton either with retention or inversion of configuration [3,4]. These polysaccharides may also be modified by the action of lyases. Thus, in the modification of alginic acid, a ‘mannuronate lyase’ (alginate lyase, EC 4.2.2.3) degrades the polymer to oligosaccharides containing 4-deoxy-L-erythro-hex-4-ene pyranosyluronate at the non-reducing end. Gacesa [5] has proposed an extension of the carbanion mechanism to the lyase enzymes. For the lyase

Partial purification and properties of UDPG dehydrogenase from Escherichia coli

Abstract 1. 1. UDPG dehydrogenase (UDPG: NAD oxidoreductase, EC 1.1.1.22), was purified 88-fold to a specific activity of 2.3 from a derepressed strain of Escherichia coli. 2. 2. The enzyme has a molecular weight of approximately 86 000 and a pH optimum of 9.0; Km is 1.0 mM for UDPG and 0.05 mM for NAD, respectively. 3. 3. UDPG, CDPGlc and dTDPGlc, but not GDPGlc or ADPGlc function as substrates. 3-Acetylpyridine adenine dinucleotide, 3-pyridinealdehydeadenine dinucleotide, thionicotinamide adenine dinucleotide, deamino adenine dinucleotide and 3-acetylpyridine deamino adenine dinucleotide, but not 3-pyridinealdehyde deaminoadenosine dinucleotide, are cosubstrates. 4. 4. The enzyme is stable for 2 months when frozen at −4 °C in the presence of UDPG. UDPG and 2-mercaptoethanol are necessary for optimal enzyme activity. 5. 5. UDPXyl is an inhibitor strictly competitive with UDPG and noncompetitive with NAD; NADH is an inhibitor strictly competitive with NAD and uncompetitive with UDPG. UDPglucuronate is an inhibitor competitive with UDPG at high UDPG concentrations; at low UDPG concentrations UDPglucuronate is an inhibitor which displays positive cooperativity.

Biosynthesis of heparin. Loss of C-5 hydrogen during conversion of d-glucuronic to l-iduronic acid residues

Abstract Microsomal fraction from mouse mastocytoma was incubated with UDP-N-acetylglucosamine and 3 H-, 14 C-labeled UDP-glucuronic acid. Enzymatic sulfation of the resulting heparin-precursor polysaccharide (by addition of 3′-phosphoadenylylsulfate) was accompanied by selective loss of 3 H from C5, but not from C2 or C4, of the uronic acid residues. Analysis of the sulfated product showed C5- 3 H in the glucuronic acid but not in the iduronic acid component. The formation of l -iduronic acid residues by C5-epimerization of d -glucuronic acid units at the polymer level thus involves loss of hydrogen at C5.

Pyridine nucleotide-linked four-electron transfer dehydrogenases

Abstract Three types of four-electron transfer, pyridine nucleotide-linked dehydrogenases are known: β-methyl-β-hydroxyglutaryl CoA reductase, histidinol dehydrogenase, and nucleoside diphosphate sugar dehydrogenases. Although the reactions catalysed by the latter two classes of enzyme are very similar, the enzymes are structurally different.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.

UDP-glucose dehydrogenase from Escherichia coli. Purification and subunit structure.

UDPglucose dehydrogenase from Escherichia coli has been purified 330-fold with an overall yield of 27%. A single homogeneous subunit was demonstrated by ultracentrifugation in 6 M guanidium chloride and by dodecyl sulfate-polyacrylamide gel electrophoresis. Since the molecular weight of the intact dehydrogenase is in the order of 86 000 and the subunit weight determined by the dodecyl sulfate-polyacrylamide gel electrophoresis is 47 000, the enzyme consists of two polypeptide chains. The sole amino terminal acid shown by the dansylation technique was arginine. Forty-four tryptic peptides were obtained by peptide mapping, in agreement with the number of arginine and lysine residues/mole protein [43] determined by amino acid analysis. The data are consistent with the presence of two identical or very similar polypeptide chains in E. coli UDPglucose dehydrogenase.

INTERFERON PRODUCTION IN MICE BY CELL WALL MUTANTS OF SALMONELLA TYPHIMURIUM. III. ROLE OF LIPID MOIETY OF BACTERIAL LIPOPOLYSACCHARIDE IN INTERFERON PRODUCTION IN ANIMALS

The interferon response elicited by Salmonella typhimurium mutants in mice is not dependent on the presence of a complete cell wall lipopolysaccharide. In fact, a mutant (G30/C21) which has lost all the polysaccharide side chains and sugars of the O antigen and contains only 2-keto-3-deoxyoctonate and lipid is indistinguishable in its interferon-stimulating ability from the wild type which possesses a complete O antigen with polysaccharide side chains.