Dev Mangroo

Phospholipid synthesis: effects of solvents and catalysts on acylation

Abstract The peptide dinitrophenylprolylthreoninamide (DNP-Pro-Thr-NH2) was used as a model system to develop better acylation conditions for the synthesis of phospholipids using catalyst-activated anhydrides. The acylation rate was found to be inversely related to the polarity of the solvent, chloroform alone resulting in much better rates of reaction than did pyridine, dimethylformamide (DMF) or mixtures of these solvents. Anhydride activated by 4-pyrrolidinopyridine (PPY) was twice as reactive as that activated by 4-dimethylaminopyridine (DMAP). It was shown that the phosphate group of phosphatidylcholine (PC) interferes with the acylation by a process which could be reserved by means of the addition of a 200-fold excess of PPY. This reversal is not due to base catalysis by the PPY; the results suggest that a mixed anhydride may be formed with the phosphate and that this can be reversed by high catalyst concentrations to produce the reactive acylating agent. The acylation rates for lysophosphatidylcholine (lyso PC) using our optimum conditions were found to be approximately 50 times faster than the best rates reported in the literature, the reaction being complete within 5 min even using only a slight excess of anhydride. acyl group migration was assessed during these reactions and no increase in migration of the acyl groups could be detected due to these reaction conditions. The procedures described provide significant improvements over previous methods described for large scale, as well as highly radioactive microscale phospholipid synthesis.

Schizosaccharomyces pombe, unlike Saccharomyces cerevisiae, may not directly regulate nuclear-cytoplasmic transport of spliced tRNAs in response to nutrient availability

Eukaryotic cells adapt to changes in nutrient levels by regulating key processes, such as gene transcription, ribosome biogenesis, and protein translation. Several studies have shown that nuclear export of tRNAs is also regulated in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress. However, recent studies suggest that nutrient stress does not affect nuclear tRNA export in several mammalian cell lines, including rat hepatoma H4IIE. Furthermore, in contrast to previous studies, data reported more recently established that nuclear export of mature tRNAs derived from intron-containing pre-tRNAs, but not mature tRNAs made from intronless precursors, is affected by nutrient stress in several species of Saccharomyces, but not in the yeast Kluyveromyces lactis . Here, we provide evidence suggesting that Schizosaccharomyces pombe, like mammalian cells and K. lactis, but unlike Saccharomyces, do not directly regulate nuclear export of mature tRNAs made from intron-containing pre-tRNAs in response to nutrient stress. These studies collectively suggest that regulation of nuclear export of spliced tRNAs to the cytoplasm in response to nutrient availability may be limited to the genus Saccharomyces, which unlike other yeasts and higher eukaryotes produce energy for fermentative growth using respiration-independent pathways by downregulating the citric acid cycle and the electron transport chain.

Specific labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives

The labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives was investigated. Proteins having molecular masses of 70 kDa, 48 kDa and 15 kDa were labeled with 11-m-diazirinophenoxy-[11-3H]undecanoate while 11-m-diazirinophenoxy-[11-3H]undecanoyl-CoA labeled proteins of 70 kDa and 55 kDa. The 70 kDa protein labeled with both photoreactive probes was resolved into two bands by electrophoresis on a gradient polyacrylamide gel; immunoprecipitation with anti-fatty acyl-CoA oxidase showed that these proteins are fatty-acyl-CoA oxidases. In purified peroxisomal membranes, two proteins of 36 kDa and 25 kDa were labeled with the photoreactive fatty-acid probe, whereas very little labeling of the above proteins or other proteins was observed with the fatty-acyl-CoA probe. The photoaffinity labeling method described is, thus, clearly capable of identifying and distinguishing between proteins having an affinity for fatty acid or fatty-acyl-CoA. The labeling also identified a fatty-acid-binding site on the 16 kDa peroxisomal matrix protein as well as on two peroxisomal acyl-CoA oxidases. This approach thus provides a general means for the identification of fatty-acid metabolizing enzymes, as well as for the identification of fatty-acid-binding sites on known enzymes.