scholarly journals ISOLATION OF HEXOKINASE FROM BAKER'S YEAST

1946 ◽  
Vol 29 (6) ◽  
pp. 379-391 ◽  
Author(s):  
Louis Berger ◽  
Milton W. Slein ◽  
Sidney P. Colowick ◽  
Carl F. Cori
Keyword(s):  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Protective Action ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Thiol Compounds ◽  
Protective Proteins ◽  
Yeast Hexokinase

1. A method is described for the isolation of hexokinase from baker's yeast. The method is based mainly on fractionation with alcohol and results See PDF for Structure in a 30-fold increase in specific activity. The final product could be crystallized from ammonium sulfate without change in specific activity. 2. The enzyme catalyzes a transfer of phosphate from adenosinetriphosphate to glucose, fructose, or mannose, the relative rates with these three sugars being 1:1.4:0.3. 3. With glucose as substrate, the turnover number for the crystalline enzyme is 13,000 moles of substrate per 105 gm. of protein per minute at 30° and pH 7.5. The temperature coefficient (Q10°) between 0 and 30° is 1.9. 4. Magnesium ions are necessary for the activity, the dissociation constant for the Mg++ -protein complex being 2.6 x 10–3. Fluoride in concentrations as high as 0.125 M has no inhibitory effect on the enzyme when the Mg++ and orthophosphate concentrations are 6.5 x 10–3 M and 1 x 10–3 M, respectively. 5. The crystalline enzyme shows a loss in activity when highly diluted. This loss in activity can be prevented by diluting in the presence of small amounts of other proteins. Of the various protective proteins tested, insulin was the most effective, providing complete protection in a concentration of 6 micrograms per cc.; with serum albumin, a concentration of 60 micrograms per cc. was necessary. Thiol compounds (cysteine, glutathione) exerted no protective action. 6. The inactivation of the crystalline enzyme on incubation with trypsin can be prevented to a marked degree by the presence of glucose. The instability of crude preparations of yeast hexokinase may be attributed to the presence of proteolytic enzymes, since glucose or fructose has a remarkable protective effect on such preparations.

Phenylalanine Analogues: Potent Inhibitors of Phenylalanine Ammonia-Lyase Are Weak Inhibitors of Phenylalanine-tRNA Synthetases

1994 ◽  
Vol 49 (11-12) ◽  
pp. 781-790 ◽  
Author(s):  
Gerhard Leubner Metzger ◽  
Nikolaus Amrhein
Keyword(s):  
Wheat Germ ◽  
Phenylalanine Ammonia Lyase ◽  
Inhibitory Effect ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Exchange Reactions ◽  
Trna Synthetases ◽  
Different Sources

(1-Amino-2-phenylethyl)phosphonic acid (APEP), (1-amino-2-phenylethyl)phosphonous acid (APEPi), α-aminooxy-β-phenylpropionic acid (AOPP) and several other phenylalanine analogues are potent inhibitors of (S)-phenylalanine ammonia-lyase (PAL) in vitro and in vivo. The ability of these compounds to inhibit (S)-phenylalanine-tRNA synthetases (PRSs) from wheat germ, soybean, and baker’s yeast has been investigated and compared to the inhibition of PAL. APEP and APEPi were found to inhibit the tRNAphe-aminoacylation reactions catalyzed by the three PRSs studied in vitro in a competitive manner with respect to (5)-phenylalanine. (R)-APEP inhibits the PRSs with apparent Ki values of 144 μᴍ for wheat germ (app. Km for (S)-phe 5.2 μᴍ) , 130 μᴍ for soybean (app. Km for (S)-phe 0.9 μᴍ) , and 1096 μᴍ for baker’s yeast (app. Km for (S)-phe 5.5 μᴍ ) . The apparent Ki values for (R)-APEPi are 315 μᴍ , 160 μᴍ , and 117 μᴍ , respectively. APEP and APEPi inhibit the ATPpyrophosphate exchange reactions catalyzed by the PRSs from wheat germ and baker’s yeast, but they are not activated and do not serve as substrates in these reactions. AOPP has no affinity to any of the three PRSs, whereas it is a potent inhibitor of PAL. In light of our in vitro results with PRSs from different sources it appears unlikely that the PAL inhibitors we have studied have any significant inhibitory effect on this essential step in protein synthesis in vivo.

scholarly journals Exo-β-glucanases in yeast

1968 ◽  
Vol 109 (3) ◽  
pp. 347-360 ◽  
Author(s):  
Ahmed T. H. Abd-El-Al ◽  
H. J. Phaff
Keyword(s):  
Specific Activity ◽  
Culture Fluid ◽  
Competitive Inhibitor ◽  
Cell Extract ◽  
Baker’S Yeast ◽  
Maximal Velocity ◽  
Baker's Yeast ◽  
Molecular Weights ◽  
Hansenula Anomala ◽  
Single Enzyme

1. A number of yeast species were examined for the presence of β-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher β-(1→3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-β-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-β-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-β-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5·5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-β-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-δ-lactone gave the same value of Ki when tested with either substrate; (iii) quantitative application of the ‘mixed-substrate’ method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-β-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and Km values with laminarin and pustulan differed markedly. Comparison of Vmax. and Km values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-β-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.

scholarly journals Inhibitory Properties of Aqueous Ethanol Extracts of Propolis on Alpha-Glucosidase

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Hongcheng Zhang ◽  
Guangxin Wang ◽  
Trust Beta ◽  
Jie Dong
Keyword(s):  
Aqueous Ethanol ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Postprandial Hyperglycemia ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Ethanol Extracts ◽  
Alpha Glucosidase ◽  
Mammalian Intestine ◽  
Intestinal Sucrase

The objective of the present study was to evaluate the inhibitory properties of various extracts of propolis on alpha-glucosidase from baker’s yeast and mammalian intestine. Inhibitory activities of aqueous ethanol extracts of propolis were determined by using 4-nitrophenyl-D-glucopyranoside, sucrose and maltose as substrates, and acarbose as a positive reference. All extracts were significantly effective in inhibitingα-glucosidase from baker’s yeast and rat intestinal sucrase in comparison with acarbose (P<0.05). The 75% ethanol extracts of propolis (75% EEP) showed the highest inhibitory effect onα-glucosidase and sucrase and were a noncompetitive inhibition mode. 50% EEP, 95%, EEP and 100% EEP exhibited a mixed inhibition mode, while water extracts of propolis (WEP) and 25% EEP demonstrated a competitive inhibition mode. Furthermore, WEP presented the highest inhibitory activity against maltase. These results suggest that aqueous ethanol extracts of propolis may be used as nutraceuticals for the regulation of postprandial hyperglycemia.

Substrate specificity and some other properties of baker's yeast hexokinase

1958 ◽  
Vol 30 (1) ◽  
pp. 92-101 ◽  
Author(s):  
Alberto Sols Gertrudis De la Fuente ◽  
Carlos Villar-Palasí ◽  
Carlos Asensio
Keyword(s):  
Substrate Specificity ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Yeast Hexokinase

scholarly journals Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker's yeast

1969 ◽  
Vol 112 (5) ◽  
pp. 755-762 ◽  
Author(s):  
J. J. Cazzulo ◽  
A. O. M. Stoppani
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Carboxylase ◽  
Physiological Role ◽  
Inhibitory Effect ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Acetyl Coa ◽  
Competitive Inhibitors ◽  
Adenosine Phosphates ◽  
Regulation Of Enzyme Activity

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8·0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD+, NADP+ and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD+, NADH, NADP+ and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6·5, in the presence of 12mm-Mg2+, 0·75mm-Mn2+ and 0·5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.

scholarly journals The purification and some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast

1972 ◽  
Vol 126 (1) ◽  
pp. 27-34 ◽  
Author(s):  
B. Middleton ◽  
P. K. Tubbs
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Reaction Pathway ◽  
Gel Filtration ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Complete Protection ◽  
Acetyl Coa ◽  
Molecular Weights ◽  
Enzyme Intermediate

1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (μmol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5μm, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10–30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.

scholarly journals S-adenosylmethionine decarboxylase from baker's yeast

1975 ◽  
Vol 151 (1) ◽  
pp. 67-73 ◽  
Author(s):  
H Pösö ◽  
R Sinervirta ◽  
J Jänne
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Baker’S Yeast ◽  
Decarboxylase Activity ◽  
Spermidine Synthase ◽  
Baker's Yeast ◽  
Purification Procedure ◽  
Adenosylmethionine Decarboxylase ◽  
Km Value

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1′[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5′-deoxyadenosyl-(5′),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5′-phosphate.

scholarly journals Effect of temperature, concentration of alcohols and time on baker’s yeast permeabilization process

2018 ◽  
Vol 3 (21) ◽  
pp. 195-206
Author(s):  
Ilona Trawczyńska ◽  
Justyna Miłek ◽  
Sylwia Kwiatkowska-Marks
Keyword(s):  
Fold Increase ◽  
Alcohol Concentration ◽  
Baker’S Yeast ◽  
Yeast Cells ◽  
Process Efficiency ◽  
Effect Of Temperature ◽  
Baker's Yeast ◽  
Traditional Use ◽  
Intracellular Enzyme ◽  
Lower Alcohol

Baker’s yeast beyond the traditional use in the food industry may be used to carry out biotransformations. The effectiveness of yeast as biocatalysts is based on the presence of large amounts of intracellular enzymes, whose efficiency can be repeatedly increased by permeabilization. It is the process of increasing the permeability of cell walls and membranes in order to facilitate reagents access to the intracellular enzyme. Alcohols permeabilization process allows for approx. 50-fold increase in catalase activity of baker’s yeast. In this paper, the influence of physical and chemical parameters on the effectiveness of permeabilization of baker’s yeast cells using alcohols was analyzed. Research has shown that with increasing temperature of permeabilization process better results are achieved using a lower alcohol concentration. Based on presented response surface graphs, we can also indicate a negligible impact of duration time on the process efficiency.

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