RESUMO
Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for delta-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16.2 mumol h-1 per mg protein at pH 9.4 and 37.5 degrees C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr of 275,000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr congruent to 37,000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1.0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total delta-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.
Assuntos
Sintase do Porfobilinogênio/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Plasmídeos , Porfobilinogênio/metabolismo , Sintase do Porfobilinogênio/genética , Sintase do Porfobilinogênio/metabolismo , Protaminas , Saccharomyces cerevisiae/genética , Compostos de Sulfidrila , Temperatura , Transformação Genética , Zinco/farmacologiaRESUMO
Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d.
Assuntos
Citocromos/biossíntese , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Heme/biossíntese , Saccharomyces cerevisiae/genética , 5-Aminolevulinato Sintetase/biossíntese , Aerobiose , Desoxiglucose/farmacologia , Indução Enzimática/efeitos dos fármacos , Retroalimentação , Genes Fúngicos , Glucose/farmacologia , Maltose/farmacologia , Sintase do Porfobilinogênio/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Saccharomyces cerevisiae mutants bearing mutations at the cyc4 locus are partially deficient in cytochrome synthesis. Although the mutation is not in the structural gene for delta-aminolevulinic acid (Alv) synthase, the mutants are deficient in Alv synthesis in vivo as indicated by abnormally low intracellular Alv concentrations. The cyc4 mutation causes cells to grow very slowly in minimal glucose medium, but not in yeast extract-peptone-glucose medium. A simple nutritional defect caused by the cyc4 mutation is not involved because cytochrome deficiency is enhanced by growing cyc4 cells in yeast extract-peptone medium. A regulatory role for CYC4 is indicated. Evidence for negative feed-back control of Alv synthase by heme is provided by the observation of enhanced intracellular Alv accumulation in yeast mutants partially deficient in decarboxylation of uroporphyrinogen and coproporphyrinogen, respectively.