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t-Butyl hydroperoxide has been studied as a possible competitive inhibitor of the vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluIle. Under standard carboxylating conditions the concentrations of reduced phylloquinone and phylloquinone were followed by high-pressure liquid chromatography during 30-min incubations of Triton-solubilized microsomes from rat liver. Under these conditions supporting linear rates of carbon dioxide fixation for 20–30 min, the vitamin KH2 concentration decreased exponentially to less than 5% of its initial value in 30 min principally due to autooxidation. In the presence of 10 m m t-butyl-OOH, however, the oxidation of vitamin KH2 was greatly accelerated with none being detected after 7 min. In general, the rate of carboxylation of peptide paralleled the KH2 concentration. After cessation of carboxylation in the presence of t-butyl-OOH the readdition of KH2 stimulated additional 14CO2 fixation. A known competitive inhibitor of vitamin K, 2-chlorophylloquinone, did not accelerate the oxidation of KH2 but nonetheless inhibited the vitamin K-dependent carboxylation in a competitive manner. These data have led us to conclude that t-butyl-OOH is not a competitive inhibitor of the vitamin K-dependent carboxylase at the active site of the enzyme but merely acts to promote the oxidation of KH2.

The alkylation of menaquinone-0 to menaquinones occurs in the microsomal fraction of chick and rat liver. Subfractionation of chick microsomes into smooth and rough reticulum was achieved by density gradient centrifugation. The alkylation enzyme was localized in the smooth reticulum with an activity of 155 pmol/mg per h, about 50-fold greater than that in the rough reticulum. The membrane-bound alkylating enzyme system requires reduced menaquinone-0 and is phospholipid-dependent.

Synthetic lysophosphatidylethanolamine (LPE) obtained from commercial sources augmented the apparent activity of phospholipase A2 (PLA2) in cardiac mitochondrial and microsomal fractions. For elucidation of this phenomenon, 2-[1-14C]linoleylphosphatidylethanolamine was incubated in the absence of cell protein with selected concentrations of LPE, lysophosphatidylcholine (LPC), Ca2+, ionic and nonionic detergents, and phospholipids with functional groups similar to those of LPE. Hydrolysis of phosphatidylethanolamine (PE) was evaluated by measurement of 14C-labeled free fatty acid release and confirmed by quantification of [14C]ethanolamine-labeled LPE formed. The reaction was dependent on the concentrations of Ca2+, PE, and LPE, exceeding 1.5 nmol/h with 20 micro M LPE and 30 micro M PE. Hydrolysis occurred in the presence of as little as 1 micro M LPE. PE was not hydrolyzed by comparable concentrations of ionic or nonionic detergents or by several closely related phosphatides, including LPC. Purification of synthetic LPE by high-performance LC to remove contaminating PLA2 eliminated the effect. LPE-induced hydrolysis of PE was found to depend on contamination of the LPE by PLA2 from Crotalus atrox, employed in the commercial synthesis of the lysophosphatide from the precursor used, phosphatidylethanolamine. Contamination of commercially obtained lysophosphoglycerides by PLA2 constitutes a technical pitfall which may cloud interpretation of experiments performed with inadequately purified material.

Publisher Summary Saccharomyces cerevisiae αS288C is a strain of baker's yeast that is widely used for genetic studies. This well-characterized organism is suitable for the preparation of both tRNA and amino acid:tRNA ligases. This organism has been strongly recommended, or an isogenic strain, for all biochemical studies involving yeast tRNA and its related enzymes. This chapter describe the preparation of homogeneous amino acid:tRNA ligases specific for tyrosine, alanine, ratine, and isoleucine from Saccharomyces cerevisiae αS288C. These ligases are the first to be reported from this organism. It is believed that the procedures described will prove generally applicable for most, if not all, of the amino acid:tRNA ligases from this organism. The chapter discusses the properties of these enzymes. The molecular weight and subunit structure of the amino acid:tRNA ligases are determined under denaturing conditions by discontinuous polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate.