No abstract

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes,slr1736 and HPT1, that encode HPT fromSynechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystissp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.

Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.

L-myo-Inositol-1-phosphate synthase has been purified to homogeneity from bovine testis by (NH4)2SO4 precipitation on Celite followed by reverse (NH4)2SO4 gradient elution, DEAE chromatography, gel filtration, and hydroxylapatite chromatography. The enzyme is then pure by the criteria of elution profile from the hydroxylapatite, electrophoresis, and sedimentation properties. We find no overall (gluconeogenic) reversibility of the enzyme using 6 mM DL-myo-inositol-1-P. The first three steps of the reaction are reversible as determined by uptake of isotope from a D2O incubation medium into the 6 position of D-glucose-6-P. Thus, substrate binding, dehydrogenation, and proton removal prior to the aldol cyclization are all reversible steps. The enzyme is less than 5% NAD+ independent and is not inhibited by substrate or product (5 mM D-glucose-6-P or 0.8 mM DL-myo-inositol-1-P). The enzyme is twofold stimulated by either 50 mM NH4+ or 50 mM K+; the activation by these ions is not additive. Sodium ions inhibit the enzyme by 78% at 153 mM. The effect of sodium and potassium is not on the Km of D-glucose-6-P but on Vmax. We propose that K+ activates the enzyme by stabilizing a carbanion intermediate. Ethanol stimulates the enzyme 2-fold and 2.5-fold with added K+. The effect of ethanol appears to be via lowering of the D-glucose-6-P Km. In the presence of ethanol the effect of salt on Vmax disappears.

Twenty hours following the subcutaneous administration of 5 mEq/kg doses of 6LiCl and 7LiCl to two groups of rats, the cerebral cortex molar ratio of 6Li+/7Li+ is 1.5. The effects of the lithium isotopes on cortex myo-inositol and myo-inositol-l-phosphate levels are the same as we have reported earlier: a Li+ concentration-dependent lowering of myo-inositol and increase in myo-inositol-1-phosphate. Thus 6LiCl, when administered at the same dose as 7LiCl, produces the larger effect on inositol metabolism. When the 6LiCl and 7LiCl doses were adjusted to 5 mEq/kg and 7 mEq/kg, respectively, the cortical lithium myo-inositol and myo-inositol-1-phosphate levels of each group of animals became approximately equal, suggesting that the isotope effect occurs at the level of tissue uptake, but not on inositol phosphate metabolism. The inhibition of myo-inositol-1-phosphatase by the two lithium isotopes in vitro showed no differential effect. The isotope effect on cerebral cortex uptake of lithium is in the same direction as that reported by others for erythrocytes and for the CSF/plasma ratio, but of larger magnitude.