scholarly journals Acid phosphatase of the yeast Saccharomyces cerevisiae. Purification and properties of the enzyme

2015 ◽  
Vol 46 (2) ◽  
pp. 173-186 ◽  
Author(s):  
W. Wątorek ◽  
B. Morawiecka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fluoride Ions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purification And Properties ◽  
Total Molecular Weight ◽  
C 50 ◽  
Sepharose 4B

Acid phosphatase from the yeast <i>Saccharomyces cerevisiae</i> was purified to homogeneity as ascertained by ultracentrifugation and electrophoresis. The purification procedure involved mechanical cell disruption, ethanol precipitation, chromatography on DEAE-cellulose, gel filtration on Sepharose 4B. The sedimentation constant S20<sup>0.580</sup> of the purified enzyme was 15.4 S. Carbohydrate content accounted for 50% of the total molecular weight of the enzyme. The optimum pH for purified enzyme was 3.0-3.5, it was stable at pH 3.0-5.0 at room temperature. After 10 min. incubation at 45° C, 50 per cent of the enzymatic activity was lost. Michaelis constant was found to be 1.3 x 10<sup>-4</sup> M for p-nitrophenylphosphate and 5 x 10<sup>-4</sup> M for 3-glycerophosphate as substrates. The enzyme was inhibited by Hg<sup>2+</sup>, Cu<sup>2+</sup>, Fe<sup>3+</sup>, molybdate, phosphate, arsenate, fluoride ions. Inhibition caused by fluoride ions was noncompetitive, by phosphate - competitive, 5 M urea inactivated the enzyme completely, inactivation was reversible at urea concentration below 2,5 M.

scholarly journals Purification and properties of acid phosphatase from Avena elatior L. seeds

2015 ◽  
Vol 46 (3) ◽  
pp. 481-488 ◽  
Author(s):  
E. Wieczorek ◽  
I. Lorenc-Kubis ◽  
B. Morawiecka
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Sepharose 4B

Acid phosphatase F1 from <i>Avena elatior</i> seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn<sup>2+</sup>, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

scholarly journals Purification and properties of α-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells

1997 ◽  
Vol 324 (3) ◽  
pp. 951-956 ◽  
Author(s):  
Jianxin REN ◽  
Francis J. CASTELLINO ◽  
Roger K. BRETTHAUER
Keyword(s):  
Spodoptera Frugiperda ◽  
Reaction Pathway ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Mannose Residue ◽  
Lepidopteran Insect ◽  
Reducing Conditions ◽  
Apparent Homogeneity ◽  
Purification And Properties

An α-mannosidase II-like activity was identified in baculovirus-infected Spodoptera frugiperda (IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) and p-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+ was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

1995 ◽  
Vol 42 (2) ◽  
pp. 269-274 ◽  
Author(s):  
U Lenart ◽  
J Haplova ◽  
P Magdolen ◽  
V Farkas ◽  
G Palamarczyk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Deae Cellulose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sds Page ◽  
Nonionic Detergent ◽  
Membrane Bound ◽  
Labeled Probe ◽  
Triton X ◽  
Cellulose Column

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

scholarly journals Purification and properties of a new testosterone 17β-dehydrogenase (NADP+) from guinea-pig liver

1977 ◽  
Vol 163 (3) ◽  
pp. 401-407 ◽  
Author(s):  
E Kaguera ◽  
S Toki
Keyword(s):  
Guinea Pig ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Pig Liver ◽  
Ring Junction ◽  
Purification And Properties ◽  
Androstane Derivatives ◽  
Guinea Pig Liver

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.

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 Identification of coated vesicles in Saccharomyces cerevisiae.

1984 ◽  
Vol 98 (1) ◽  
pp. 341-346 ◽  
Author(s):  
S C Mueller ◽  
D Branton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Filtration ◽  
Coated Vesicles ◽  
Yeast Cells ◽  
Coated Vesicle ◽  
Contour Length ◽  
Yeast Saccharomyces Cerevisiae ◽  
Negative Stain ◽  
Sds Page ◽  
Negative Stain Electron Microscopy

Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The presence of coated vesicles in yeast cells suggests that this organism will be useful for studying the function of clathrin-coated vesicles.

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