Protease B from Saccharomyces cerevisiae. Purification and characterization.

Protease B has been isolated from Saccharomyces cerevisiae and purified in six steps as follows: autolysis of the yeast cells, ammonium sulfate fractionation, activation of the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown to be homogeneous on polyacrylamide gels in the absence as well as in the presence of sodium dodecylsulfate. Furthermore, the molecular weight (43,000 daltons) and the isoelectric point (5.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence of disulfide bonds in protease B has to be outlined. The amino acid residues of the protein have been found to be folded nearly quantitatively (at least 80%) in a beta-conformation as deduced from a circular dichroism study. Finally, the tryptophan residues (5 mol/mol protein) are largely buried in the hydrophobic core of the enzyme.     

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.     

Isolation and characterization of valine transfer RNA from Saccharomyces cerevisiae.

Two procedures for isolating valine tRNA from commercial bakers' yeast were investigated. The first involved: (a) counter double current distribution; (b) chromatography on benzoyl-DEAE-cellulose; (c) reverse phase chromatography on Chromosorb G saturated with trioctylpropylammonium bromide (Oakridge System 3). The material isolated lacked the 3'-terminal adenylic acid residue. The second procedure involved the first two steps above followed by: (a) enzymatic aminoacylation with a partially purified yeast extract; (b) derivatization with N-phenoxyacetoxysuccinimide; (c) chromatography on benzoyl-DEAE-cellulose; (d) reverse phase chromatography, System 3. The product was intact tRNA. It was a mixture of isoacceptors (59:41) differing by a modification (uracil leads to dihydrouracil) at position 48. It was free of denatured material; specific activity 1,825 pmol of valine/A260 unit of tRNA. Sequence analysis confirmed the recently corrected structure (Bonnet, J., Ebel, J. P., Dirheimer, G., Shershneva, L. P., Krutilina, A. I., Venkstern, T. V., and Bayev, A. A. (1974) Biochimie 56, 1211-1213). A preliminary study of the alkaline hydrolysis of the 7-methylguanosine residue that occurs at position 47 showed that at least two products are formed instead of only one as usually quoted in the literature. A rapid, ultramicro, chromatographic system for separating these products and measuring them quantitatively is described.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.

Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).     

Cloning and characterization of a cysteine proteinase from Saccharomyces cerevisiae.

We have isolated a gene from Saccharomyces cerevisiae that encodes a protein homologous to the mammalian cysteine proteinase bleomycin hydrolase. Sequence comparison between the yeast and rabbit proteins indicates an amino acid identity of 41.5% over 277 residues and a similarity of 78.3% when conservative substitutions are included. The apparent mass of the yeast protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 47 kDa, although sequence analysis indicates two potential initiator methionines that suggest calculated masses of either 51 or 55 kDa. The protein is nonessential in yeast as haploid mutants disrupted at several positions along the open reading frame remain viable. Furthermore, these mutants do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients, osmotic strength, or exogenous bleomycin. However, the purified protein does exhibit marked hydrolytic activity toward the substrate arginine 4-methyl-7-coumarylamide (Km = 12.8 microM, Vmax = 2.56 mumol mg-1 h-1), and yeast cells engineered to express this protein at higher levels maintain increased resistance to bleomycin compared to wild-type cells. Because this protein represents the first example of a cysteine proteinase identified in yeast, we have named it Ycp1 (yeast cysteine proteinase).     

DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.     

Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was purified 12 000-fold to homogeneity from yeast by a three-step procedure including acid precipitation, anion-exchange chromatography, and guanosine 5' -monophosphate affinity chromatography. The enzyme is a dimer consisting of two, probably identical, subunits of Mr 29 500. The enzyme recognized hypoxanthine and guanine, but not adenine or xanthine, as substrates. An antiserum against both native and denatured enzyme has been raised and shown to be specific for the enzyme. The antiserum has no affinity for Chinese hamster or human HPRT but does recognize subunits of yeast HPRT as well as some cyanogen bromide fragments of the enzyme.     

The purification and characterization of arginase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.     

Cloning and characterization of methenyltetrahydrofolate synthetase from Saccharomyces cerevisiae

The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.     

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.