Pyruvate Oxidase Activity Research Articles

Lipid activation and protease activation of pyruvate oxidase. Evidence suggesting a common site of interaction on the protein.

Lipid activation and protease activation of pyruvate oxidase. Evidence suggesting a common site of interaction on the protein.

Characterization of the proteolytic activation of pyruvate oxidase. Control by specific ligands and by the flavin oxidation-reduction state.

Characterization of the proteolytic activation of pyruvate oxidase. Control by specific ligands and by the flavin oxidation-reduction state.

Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids.

The pyruvate oxidase system of Escherichia coli is composed of a soluble flavoprotein, pyruvate oxidase (EC 1.2.2.2, pyruvate:ferricytochrome b1 oxidoreductase), and an electron transport system associated with the cell envelope-membrane fraction. The membrane particles contain 15% lipid by weight. Fractionation of the lipids revealed that abut one-third are neutral lipids and two-thirds are phospholipids. The relative ratio of ubiquinone to menaquinone within the neutral lipid fraction is 15:1 on a molar basis. Removal of the lipids from the membrane particles by extraction with aqueous acetone or hydrolysis of the phospholipids by treatment with Bacillus cereus phospholipase C results in a complete loss of electron transport activity. Analysis of the particles extracted with aqueous acetone revealed that practically all the neutral lipids and 65% of the phospholipids are removed by this treatment. Phospholipase treatment results in a loss of 75% of the membrane phospholipid phosphorus; however, the diglycerides and the neutral lipids produced by phospholipase hydrolysis remain associated with the particles. Addition of neutral lipid and a detergent, hepta-DL-alanyl dodecylamide to the acetone-extracted material results in a restoration of 37% of the original particle activity. Addition of neutral lipid and hepta-DL-alanyl dodecylamide to phospholipase-treated particles completely restores the original electron transport activity. Furthermore, addition of ubiquinone from either yeast (UQ6) or E. coli (UQ8) will restore pyruvate oxidase activity when the quinones are supplemented with photoinactivated neutral lipid. No restoration of activity to phospholipase-treated particles is noted upon the addition of either menaquinone 6 or menaquinone 8 to the reconstitution system. In fact, these compounds appear to suppress restoration of activity when they are added to reaction mixtures containing neutral lipid and phospholipase-treated particles.

Crystalline Pyruvate Oxidase from Escherichia coli: II. ACTIVATION BY PHOSPHOLIPIDS

Abstract The activity of pyruvate oxidase from Escherichia coli increases 15- to 20-fold in the presence of phospholipids extracted from cell membranes. Fractionation and analysis of the phosphatides from E. coli cell envelope preparations revealed that lysophosphatidylethanolamine exhibited highest specific activity for stimulating the flavoprotein if the phospholipids were added directly to the assay mixtures. However, when water-soluble micellar preparations were substituted for direct addition of the phospholipid to the assay, all the phosphatides demonstrated higher specific activities for stimulating pyruvate oxidase. Furthermore, the differences originally noted in the activating capacities of the various cell envelope phospholipids were minimized. By measuring pyruvate oxidase activity under conditions in which the micellar phospholipid preparation was rate-limiting, Michaelis constants (Km) for various phosphatides were determined. The Km values for the cell envelope phospholipids, synthetic phosphatidylethanolamine, lecithin, and lysolecithin ranged from 0.9 to 2.2 x 10-6 m. The Km value for phosphatidylserine was somewhat higher, being 6.5 x 10-6 m. The diacylphospholipids exhibited normal Michaelis-Menten kinetics. Lysophosphatides demonstrated considerable divergence from normal Michaelis-Menten kinetics. This observation suggests that pyruvate oxidase differentiates between phospholipids according to the number of fatty acids contained within the molecular structure. The hydrophobic moieties of lecithin activate pyruvate oxidase whereas the hydrophilic portions of the molecule have no stimulatory effect. The enzyme can also be activated by fatty acids at concentrations only slightly higher than those required for maximal stimulation by micellar phospholipid preparations. Of the fatty acids tested, palmitoleic and oleic appear to be most efficient in stimulating pyruvate oxidase activity.

Metabolic activities of vitamin D in animals. 3. Biogenesis of vitamin D sulfate in animal tissues.

1. The chicks were given S35-sulfate orally and intramuscularly and the radioactivities of the left tibia, keel and skin were tested. The deposition of S35-sulfate in the tissues of the birds receiving vitamin D3 was found to be higher than those receiving vitamin D2 or vitamin D-deficient group.2. Carrier-free P32-phosphate solution was supplied orally or intramuscularly, and the radioactivities of the left tibia, keel and skin were estimated. Increased levels of P32-phosphate were always seen in the groups receiving vitamin D3.3. The activity of citrate oxidase increased significantly in the group receiving vitamin D3, while no influence was observed on the ability of oxidative phosphorylation of citrate in the liver.4. No influence was observed on the pyruvate oxidative phosphorylating ability and pyruvate oxidase activity in the liver homogenate by the administration of vitamin D3.5. The glutathione level in the blood was estimated, and the increase was statistically significant in the group receiving vitamin D3. But no influence of glutathione in the liver was observed. As for the oxidized glutathione levels, no effects were seen in both blood and liver.6. From these findings, there seems to be some intimate relationship in chicks between vitamin D3 metabolism and active sulfate metabolism postulated by Lipmann.

METABOLIC ACTIVITIES OF VITAMIN D IN ANIMALS. II. ACTION OF VITAMIN D3 IN ACTIVE SULFATE METABOLISM OF CHICKS.

1. The chicks were given S35-sulfate orally and intramuscularly and the radioactivities of the left tibia, keel and skin were tested. The deposition of S35-sulfate in the tissues of the birds receiving vitamin D3 was found to be higher than those receiving vitamin D2 or vitamin D-deficient group.2. Carrier-free P32-phosphate solution was supplied orally or intramuscularly, and the radioactivities of the left tibia, keel and skin were estimated. Increased levels of P32-phosphate were always seen in the groups receiving vitamin D3.3. The activity of citrate oxidase increased significantly in the group receiving vitamin D3, while no influence was observed on the ability of oxidative phosphorylation of citrate in the liver.4. No influence was observed on the pyruvate oxidative phosphorylating ability and pyruvate oxidase activity in the liver homogenate by the administration of vitamin D3.5. The glutathione level in the blood was estimated, and the increase was statistically significant in the group receiving vitamin D3. But no influence of glutathione in the liver was observed. As for the oxidized glutathione levels, no effects were seen in both blood and liver.6. From these findings, there seems to be some intimate relationship in chicks between vitamin D3 metabolism and active sulfate metabolism postulated by Lipmann.