PURIFICATION AND PROPERTIES OF AN INDUCIBLE β-GLUCOSIDASE OF BAKERS' YEAST

1966 ◽  
Vol 44 (8) ◽  
pp. 1099-1108 ◽  
Author(s):  
A. N. Inamdar ◽  
J. G. Kaplan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Hydrolytic Activity ◽  
Chromogenic Substrate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Intact Cells ◽  
Highly Active ◽  
Naturally Occurring ◽  
Purification And Properties ◽  
Competitive Inhibitors

The inducible β-glucosidase present in crude extracts of cellobiose-grown bakers' yeast (Saccharomyces cerevisiae C) was purified 50-fold and found to be homogeneous in the ultracentrifuge, with a molecular weight of 313,000. The enzyme was virtually identical in its properties with the internal, cryptic enzyme of the yeast cell, revealed by butanol treatment of the suspensions. It was unlike the membrane-localized enzyme found at the surface of intact cells in its low affinity for cellobiose and methyl-β-glucoside as substrates and inhibitors. The enzyme was specific for the β configuration and had no activity against substrates such as α-glucosides, β-galactosides, or β-xylosides. It was highly active against both naturally occurring and synthetic substrates with aromatic aglycones, and may thus be classed as an aryl-β-glucosidase. The enzyme had weak hydrolytic activity against methyl-β-glucoside and cellobiose, but these compounds, unlike all of the aryl-β-glucosides tested, were not competitive inhibitors of its activity against the chromogenic substrate pNPG. There were about 40,000 molecules of enzyme per cell in fully induced cultures and the enzyme represented about 3% of the total protein of these cells.

scholarly journals The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae)

1985 ◽  
Vol 231 (3) ◽  
pp. 777-779 ◽  
Author(s):  
T Dreyer ◽  
M J Valler ◽  
J Kay ◽  
P Charlton ◽  
B M Dunn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase Inhibitor ◽  
Aspartic Proteinase ◽  
Significant Extent ◽  
Aspartic Proteinases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Naturally Occurring ◽  
Proteinase A

The ability of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae) to affect the activities of a range of mammalian and microbial aspartic proteinases was examined. The inhibitor appeared to be completely selective in that only the aspartic proteinase A from yeast was inhibited to any significant extent. IA3 thus represents the first example of a totally specific, naturally occurring, aspartic-proteinase inhibitor.

scholarly journals Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.

1987 ◽  
Vol 105 (5) ◽  
pp. 1981-1987 ◽  
Author(s):  
R A Preston ◽  
R F Murphy ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Lucifer Yellow ◽  
Ph Dependence ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Low Molecular Weight ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fluid Phase Endocytosis ◽  
Rapid Kinetics

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

scholarly journals A gene with specific and global effects on recombination of sequences from tandemly repeated genes in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 711-718 ◽  
Author(s):  
R L Keil ◽  
A D McWilliams
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Copy Number ◽  
Tandem Repeats ◽  
Chromosomal Location ◽  
Specific Factor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Naturally Occurring ◽  
Type Information ◽  
Repeated Genes

Abstract The preservation of sequence homogeneity and copy number of tandemly repeated genes may require specific mechanisms or regulation of recombination. We have identified mutations that specifically affect recombination among natural repetitions in the yeast Saccharomyces cerevisiae. The rrm3 mutation stimulates mitotic recombination in the naturally occurring tandem repeats of the rDNA and copper chelatin (CUP1) genes. This mutation does not affect recombination of several other types of repeated genes tested including Ty elements, mating type information and duplications created by transformation. In addition to stimulating exchange among the multiple CUP1 repeats at their natural chromosomal location, rrm3 also increases recombination of a duplication of CUP1 units present at his4. This suggests that the RRM3 gene may encode a sequence-specific factor that contributes to a global suppression of mitotic exchange in sequences that can be maintained as tandem arrays.

Isolation and Characteristics of High-Activity Heparin

1979 ◽  
Author(s):  
Isidore Danishefsky ◽  
Steven Radoff ◽  
Michael Bender ◽  
German Villanueva
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Affinity Chromatography ◽  
High Activity ◽  
Intestinal Mucosa ◽  
Chromogenic Substrate ◽  
Highly Active ◽  
The Common ◽  
Heparin Preparation ◽  
Inhibition Of Thrombin

Chromatography of heparin from beef lung on Biogel P-100 yielded a series of fractions with different anticoagulant activities. The assays for activity of the products were based on their effect in accelerating the inhibition of thrombin by antithrombin. Residual thrombin was measured by its effect on the clotting of fibrinogen and by its action on the chromogenic substrate H-D-Phe-Pip-Arg-p-nitroanilide (S-2238). Fractions with as much as 6-10 times the clotting activity of the original heparin, were obtained. The activities per mg of heparin were highest in fractions eluted in, or near, the void volume of the column. In addition to glucosamine and uronlc acid, the most active fractions contained considerable amounts of protein. The amino acids included glycine, serine, threonine, alanine, aspartate and glutamate. Experiments with heparin from porcine intestinal mucosa gave similar results.Affinity chromatography of the highly active fractions on antithrombin-Sepharose showed that all of these materials are bound to the gel and that relatively large proportions are eluted with 1 M and 3 M NaCl.It is concluded that the common heterodisperse heparin preparation contain a series or components ranging from extremely high activities to no activity. On the basis of the elution volumes of the fractions, the activities are directly related to molecular weight.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases.

1987 ◽  
Vol 7 (12) ◽  
pp. 4390-4399 ◽  
Author(s):  
C M Moehle ◽  
R Tizard ◽  
S K Lemmon ◽  
J Smart ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Serine Proteases ◽  
Proteinase K ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Protease B ◽  
Mature Protease

The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.

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