RESUMO
Acid and neutral trehalase activities (optimum pH of 4.6 and 6.8, respectively) from Fusarium oxysporum var. lini were studied separately through partial isolation by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Sephacel for neutral enzyme, or using some of their differential properties. Acid activity was unaffected by 1 mM of Ca2+, Mg2+, Mn2+, Ba2+, or EDTA. Contrarily, the neutral enzyme was activated by Ca2+ with an apparent Ka of 0.15 mM; was inhibited by EDTA, Zn2+, Hg2+, or Mg(2+)-ATP; and showed an increase in activity by the raise of buffer ionic strength or by the addition of 100 mM KCl. Acid and neutral enzymes have, respectively, an apparent optimum temperature of 45 and 30 degrees C, an apparent Km for trehalose of 0.43 and 8.45 mM, and an apparent M(r) of 160,000 and 100,000 (by glycerol gradient ultracentrifugation). Acid trehalase was specifically inhibited by acetate buffer and more stable at 50 degrees C than the neutral enzyme. Neutral enzyme exhibited a pI of 6.2 by isoelectric focusing. Contrary to neutral trehalases from other fungi, the enzyme from Fusarium oxysporum var. lini was not activated in crude extract by treatment with Mg(2+)-ATP in the presence of cAMP and not inactivated by alkaline phosphatase from Escherichia coli.
Assuntos
Fusarium/enzimologia , Trealase/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Trealase/química , Trealase/isolamento & purificaçãoRESUMO
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
Different strains of Saccharomyces cerevisiae exhibit alternative patterns of trehalose accumulation during growth on a glucose medium. The active pattern is designated as TAC(+) phenotype and the alternative, low-activity pattern as tac(-) phenotype. The tac(-) phenotype is expressed only during growth, since tac(-) strains actively accumulate trehalose during incubation in a glucose medium lacking a nitrogen source. The tac(-) phenotype appears to be determined by a single, recessive gene tac1. The quantitative expression of the dominant, alternative allele TAC1 is subject to wide variation. A highly active pattern of trehalose accumulation requires TAC1 and an amplification factor, TAM, which consists of one or more dominant gene(s). TAM does not appear to alter significantly the expression of tac1. Highly amplified TAC(+) strains may contain a labile factor not present in a TAC(+) strain which accumulates intermediate levels of trehalose.