Expression of UDP-glucuronosyltransferase cDNA in Saccharomyces cerevisiae as a membrane-bound and as a cytosolic form.

The mouse clone UDPGTm-1 encodes a UDP-glucuronosyltransferase enzyme which was isolated from a lambda gt11 cDNA library constructed with phenobarbital-induced liver mRNA [Kimura, T., & Owens, I. S. (1987) Eur. J. Biochem. 168, 515-521]. In order to establish substrate specificity, UDPGTm-1 was inserted into the yeast vector pEVP11 and expressed in Saccharomyces cerevisiae strain AH22. Cells transformed with the expression unit pUDPGTm-1c (insert in correct orientation with respect to promoter) stably transcribe the transferase cDNA. Consistent with the presence of mRNA, pUDPGTm-1c-transformed AH22 cells synthesize a transferase protein with Mr congruent to 51,000 by Western immunoblot analysis. The membrane-bound transferase expressed in yeast in glycosylated as indicated by its enhanced electrophoretic mobility in a SDS-polyacrylamide gel following endoglycosidase H treatment and detection by Western immunoblot analysis. A survey, using 12 aglycons in an assay with microsomes from cells which express the protein, shows preferential glucuronidation of naphthol and estrone followed by p-nitrophenol. Testosterone, phenolphthalein, dihydrotestosterone, androsterone, and 4-methylumbelliferone are conjugated at an intermediate level. There is barely detectable glucuronidation of 3-hydroxy- and 9-hydroxybenzo[a]pyrene and no detectable conversion of morphine or lithocholic acid. The truncated cDNA (lacking the putative membrane-insertion signal-peptide coding sequence, but with a newly adapted translation-start codon) is ligated into pAAH5 and is expressed as a cytosolic transferase form in the protease-deficient ZA521 strain of S. cerevisiae. The Mr congruent to 51,000-52,000 is similar to that seen in microsomes from AH22 cells where the protein is presumably processed as it is inserted into the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfite oxidase activity in Thiobacillus novellus.

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate. Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities. The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate. Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity.

Purification of Thiobacillus novellus sulfite oxidase. Evidence for the presence of heme and molybdenum.

Sulfite oxidase from Thiobacillus novellus has been purified 206-fold. The enzyme reduced both ferricyanide and cytochrome c. The ferricyanide activity was 3-5% of the cytochrome c activity. During purification, the absorbance ratio of A413 nm/A280 nm showed a continual increase, suggesting the presence of heme in the T. novellus sulfite oxidase molecule. The absorption spectrum of the enzyme is very similar to that of rat liver sulfite oxidase which contains cytochrome b5 type heme. Gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate revealed a protein staining band of approximately 41,000 Da. Gel filtration chromatography of the enzyme in aqueous media indicated a molecular weight of 38,000. These results suggest that T. novellus sulfite oxidase is a monomer of approximately 40,000 Da. Moreover, analysis of the visible absorption spectrum of the purified enzyme and the co-elution of 413 and 280 nm absorbing material during high pressure liquid chromatography gel chromatography provided clear evidence for the presence of heme in T. novellus sulfite oxidase. EPR spectroscopy of the enzyme revealed a characteristic molybdenum spectrum, which was observed only in the presence of sulfite. Analysis of the T. novellus sulfite oxidase molybdenum cofactor showed a fluorescence spectrum indistinguishable from that displayed by the molybdenum cofactor of chicken liver sulfite oxidase. Therefore, it is concluded that T. novellus sulfite oxidase is a monomeric (Mr approximately 40,000) molybdohemoprotein.

Interaction of bisulfite with unsaturated fatty acids.

Sodium bisulfite reacted with unsaturated fatty acids, significantly increasing their polarity as determined by behavior on silica gel thin-layer chromatography. The ultraviolet absorption spectra of unsaturated fatty acids (due to the presence of double bonds) were abolished as a result of the reaction. Fatty acids containing more than one double bond (arachidonate, linolinate, and linoleate) reacted more rapidly with bisulfite than did oleate. When arachidonate double bonds were titrated with bisulfite there was a much larger spectral decrease with the first equivalent of bisulfite added than with each subsequent addition. Vitamin E, vitamin E nicotinate, and butylated hydroxytoluene significantly inhibited the reaction of bisulfite with unsaturated fatty acids. It is suggested that the reaction of bisulfite with unsaturated fatty acids may be a mechanism of SO2 toxicity.

Citrate synthesis from fatty acids and amino acids in rat ventral prostate.

The source of the remarkably high citrate level of prostate was investigated in prostate fragments. L-Aspartate and alpha-keto-glutarate formed oxaloacetate through transamination. Neither pyruvate nor acetate was as effective as palmitate in increasing the citrate level. The result suggested that the source of citrate in rat ventral prostate is oxaloacetate from amino acid transamination and acetyl CoA from fatty acid oxidation. Furthermore, the result indicated that carbohydrates contribute little to citrate production in ventral prostate.