In vitro and in vivo regulation of chloroplast glyceraldehydes-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. I. Purification and properties of isozymes

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) was purified from leaves of Chenopodium rubrum L. Aggregated (≥ 10⁶) and disaggregated (165 × 10³) molecular weight forms were obtained by gel filtration in the presence of NAD⁺ and NADP⁺, respectively. The disaggregated enzyme was separated into two isozymes by inverse ammonium sulphate gradient solubilization: "NADP-GPD I" was homotetrameric (subunit molecular weight 39 × 10³); "NADP-GPD II" was heterotetrameric (subunit molecular weights 39 × 10³ and 43 × 10³). Isoelectric focusing of the isozymes, both aggregated and disaggregated, revealed two isoelectric forms in each case, at 4.3 and 7.7. Chloroplast GPD was "NADP-suppressed" in crude extracts due to partial oxidation, incubation with dithioerythritol restored full activity.     

Lipid Composition of Purified Vesicular Stomatitis Viruses

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.     

A procedure for the synthesis of uridine-5'-diphospho-N-acetylglucosamine-14C

A complete procedure for the synthesis of 1-¹⁴C-glucosamine-labeled UDP-N-acetylglucosamine is described. Glucosamine is first phosphorylated with ATP and hexokinase to form glucosamine 6-phosphate. This is N-acetylated with acetic anhydride, and the product is converted to UDP-N-acetylglucosamine by incubation with a crude yeast extract. The sugar nucleotide is isolated from the incubation mixture by paper electrophoresis, and purified by paper chromatography.