The crystal structure of acidic β-galactosidase from Aspergillus oryzae.

The crystal structure of the industrially important Aspergillus oryzae β-galactosidase has been determined at 2.60 Å resolution. The Ao-β-gal is a large (985 residues) monomeric multi-domain enzyme that has a catalytic (α/β)8-barrel domain.

Membrane binding mechanism of an RNA virus-capping enzyme.

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation.

Virus-specific capping of tobacco mosaic virus RNA: methylation of GTP prior to formation of covalent complex p126-m7GMP.

In capping cellular mRNAs, a covalent GMP-enzyme intermediate leads to formation of G(5')ppp(5')N at the 5' end of the RNA, which is modified by methylation catalyzed by guanine-7-methyltransferase. Here we show that isolated membranes from tobacco mosaic virus (TMV)-infected plant or insect cells expressing TMV replicase protein p126, synthesized m7GTP using S-adenosylmethionine (AdoMet) as the methyl donor, and catalyzed the formation of a covalent guanylate-p126 complex in the presence of AdoMet. The methyl group from AdoMet was incorporated into p126, suggesting that the complex consisted of m7GMP-p126.

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient.

Biochemical and chemical characterization of phenylglyoxal bis(guanylhydrazone), an aromatic analogue of mitoguazone.

Since little has been known about the properties of aromatic analogues of the antineoplastic agent methylglyoxal bis(guanylhydrazone) (MGBG), an investigation was performed on phenylglyoxal bis(guanylhydrazone) (PhGBG). PhGBG competitively inhibited yeast adenosylmethionine decarboxylase (AdoMetDC) with a Ki of 65 microM. As compared to MGBG (Ki 0.

UDP-galactose:lactosylceramide alpha-galactosyltransferase activity in human placenta.

The activity of UDP-Gal:LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn2+ at pH 6.

Sugar analysis of glycoproteins and glycolipids after methanolysis by high-performance liquid chromatography with pulsed amperometric detection.

A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uronic acid in a single chromatographic step at the subnanomolar level.

Oxidation of glycolipids in liposomes by galactose oxidase.

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside.

Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase.

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method.

Exposure of the major human red-cell glycolipid, globoside, to galactose oxidase.

Plasma membrane glycolipids are localized at the outer leaflet of the lipid bilayer, and their carbohydrate portions are exposed to the environment. The efficiency of exposure has, however, not been known. We have been able to determine the availability of the major red cell glycolipid, globoside, to externally added galactose oxidase.