scholarly journals Characterization of an adenosine 3′:5′-cyclic monophosphate phosphodiesterase from baker's yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour

1977 ◽  
Vol 163 (3) ◽  
pp. 467-476 ◽  
Author(s):  
J Londesborough
Keyword(s):  
Ionic Strength ◽  
Cyclic Amp ◽  
Microsomal Fraction ◽  
Gel Filtration ◽  
Potassium Phosphate ◽  
Apparent Molecular Weight ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Ph Optimum

1. The 3′:5′-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.

scholarly journals Characterization of two trehalases in baker's yeast

1984 ◽  
Vol 219 (2) ◽  
pp. 511-518 ◽  
Author(s):  
J Londesborough ◽  
K Varimo
Keyword(s):  
Acetic Acid ◽  
Cyclic Amp ◽  
Concanavalin A ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Baker’S Yeast ◽  
Acidic Protein ◽  
Baker's Yeast ◽  
Ph Optimum

Trehalase activities at pH 5 (not inhibited by EDTA) and pH 7 (inhibited by EDTA) were present in the soluble fraction of disintegrated commercial baker's yeast. The pH 5 activity binds strongly to concanavalin A, is only partially salted out by saturated (NH4)2SO4, has an apparent Mr of 215000 (by gel filtration) and is an acidic protein. It has a Km of 1.4 mM, a broad pH optimum (at 40 mM-trehalose) between pH 4 and 5, and is activated by about 30% by 20-300 mM neutral salts such as KCl, NaNO3 and MnCl2. The enzyme is strongly inhibited by acetic acid/acetate buffers, with a Ki of about 15 mM-acetic acid. The pH 7 activity does not bind to concanavalin A, is salted out at 20-32% (w/v) (NH4)2SO4 and has an Mr of 170000 (by gel filtration). It is absolutely dependent on Ca2+ or Mn2+ ions (Mg2+ is ineffective) and strongly inhibited by neutral salts in the 20-100 mM range. It can be activated by treatment with MgATP in the presence of cyclic AMP. Activation decreases, but does not abolish, the Ca2+ requirement, and does not change the Km for trehalose (5.7 mM) or shift the sharp pH optimum at pH 6.7 (at 40 mM-trehalose).

scholarly journals Solubilization and other studies on adenylate cyclase of baker's yeast

1976 ◽  
Vol 159 (2) ◽  
pp. 363-370 ◽  
Author(s):  
K Varimo ◽  
J Londesborough
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Gel Filtration ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Centrifugal Forces ◽  
Wide Range ◽  
Solubilized Enzyme ◽  
Fold Activation

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30°C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000.

scholarly journals The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1972 ◽  
Vol 129 (4) ◽  
pp. 945-952 ◽  
Author(s):  
D. J. Sams ◽  
W. Montague
Keyword(s):  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Cyclic Monophosphate ◽  
Km Value

1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3′:5′-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent Km values for adenosine 3′:5′-cyclic monophosphate (cyclic AMP) of 3μm and 30μm, suggesting the presence of two activities. The pH optimum of the activity with the low Km value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low Km value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.

scholarly journals The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1969 ◽  
Vol 113 (1) ◽  
pp. 117-121 ◽  
Author(s):  
L. Stevens
Keyword(s):  
Ionic Strength ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Ribosomal Ribonucleic Acid ◽  
Number Of Binding Sites ◽  
Filtration Technique

1. The total intracellular concentrations of Na+, K+, Mg2+, spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4–60°. 5. Optimum binding occurred at about pH7·0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0·10–0·13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg2+ on equal terms for sites on the ribosomes.

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.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

Purification and properties of three endopeptidases from baker's yeast

1989 ◽  
Vol 35 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jerzy Nowak ◽  
Hsin Tsai
Keyword(s):  
Ethyl Ester ◽  
Baker’S Yeast ◽  
Molecular Forms ◽  
Acid Proteinase ◽  
Baker's Yeast ◽  
Ph Optimum ◽  
Protein Substrates ◽  
Milk Clotting ◽  
Hydrolysis Of ◽  
Milk Clotting Activity

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45 000 and 54 000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7–3.2 and casein at pH 2.4–2.8 and 5.5–6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4–6 M urea. Urea also stimulated the enzyme activity by 30–50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X–Tyr and X–Phe type. A proteinase B (PRB) preparation of approximately Mr 33 000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at −20 °C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72 000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15–Tyr16 peptide bond of the oxidized β-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at −20 °C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB. Similar to PRA, 4 M urea shifted its pH optimum for hydrolysis of protein substrates. PRY degraded apo-aminopeptidase Y much more efficiently than PRB or a PRA–PRB mixture. The possibility of PRY being a precursor form of PRA and PRB is discussed.Key words: yeast, endopeptidase, proteinase, purification.

A microbial biofuel cell with an air-breathing cathode for in vivo glucose sensing applications

2015 ◽  
Vol 7 (8) ◽  
pp. 3324-3326 ◽  
Author(s):  
Jin Wook Lee
Keyword(s):  
Glucose Sensing ◽  
Baker’S Yeast ◽  
Biofuel Cell ◽  
Baker's Yeast ◽  
Air Breathing ◽  
Sensing Applications ◽  
Microbial Biofuel

A hybrid biofuel cell employing baker's yeast and an abiotic cathode was designed and experimented.

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