Comparative study of two trehalase activities from Fusarium oxysporum var. lini.

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.

Purification of delta-aminolevulinate dehydratase from genetically engineered yeast.

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.

Partial purification and characterization of the interconvertible forms of trehalase from Saccharomyces cerevisiae.

Cryptic trehalase from Saccharomyces cerevisiae was purified about 3000-fold. The recovery of 970% of the original "activity" indicated the removal of an inhibitor of the enzyme. Active trehalase, obtained through phosphorylation of cryptic trehalase by cAMP-dependent protein kinase, was isolated by chromatography on DEAE-cellulose. A major phosphorylated protein, with an apparent Mr of 86,000, was detected after SDS-polyacrylamide gel electrophoresis. This protein band correlated exactly with the elution profile of trehalase activity and 32Pi incorporation into the enzyme on DEAE-cellulose chromatography. Partially purified active trehalase showed absolute specificity towards trehalose with an apparent Km of 4.79 X 10(-3) M. Both forms of the enzyme showed an apparent molecular weight of 160,000, by gel filtration. Centrifugation on a glycerol density gradient indicated multiple forms of trehalase-c, with Mr of 320,000, 160,000, and 80,000. After activation of each of these forms by protein kinase, a single form of trehalase-a was observed, with a Mr of 160,000. Trehalase-c appears to be a totally inactive form of the enzyme. The only mechanism of activation seems to be phosphorylation by cAMP-dependent protein kinase. When the protein kinase concentration was varied, at a fixed trehalase-c concentration, a sigmoidal activation plot was obtained. This result suggests the occurrence of multiple forms of cryptic trehalase.

Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system.

Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation. Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity. This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation. Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP. Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from [gamma-32P]ATP into protamine. In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells. Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2. Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP. Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction. Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells. This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form. The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase.

Biochemical genetics of trehalose metabolism in Saccharomyces cerevisiae.

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.