scholarly journals Purification and properties of two glyoxylate reductases from a species of Pseudomonas

1966 ◽  
Vol 101 (3) ◽  
pp. 781-791 ◽  
Author(s):  
LN Cartwright ◽  
RP Hullin
Keyword(s):  
Equilibrium Constants ◽  
Maximal Activity ◽  
High Order ◽  
Thiol Groups ◽  
Molecular Weights ◽  
Free Thiol ◽  
Purification And Properties ◽  
Keto Acids ◽  
Michaelis Constants

1. Two enzymes that catalyse the reduction of glyoxylate to glycollate have been separated and purified from a species of Pseudomonas. Their molecular weights were estimated as 180000. 2. Reduced nicotinamide nucleotides act as the hydrogen donators for the enzymes. The NADH-linked enzyme is entirely specific for its coenzyme but the NADPH-linked reductase shows some affinity towards NADH. 3. Both enzymes convert hydroxypyruvate into glycerate. 4. The glyoxylate reductases show maximal activity at pH6.0-6.8, are inhibited by keto acids and are strongly dependent on free thiol groups for activity. 5. The Michaelis constants for glyoxylate and hydroxypyruvate were found to be of a high order. 6. The reversibility of the reaction has been demonstrated for both glyoxylate reductases and the equilibrium constants were determined. 7. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.

scholarly journals Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus

1974 ◽  
Vol 144 (3) ◽  
pp. 477-486 ◽  
Author(s):  
T Ferenci ◽  
T Strøm ◽  
J R Quayle
Keyword(s):  
Equilibrium Constants ◽  
Gel Filtration ◽  
Reverse Reaction ◽  
Methylococcus Capsulatus ◽  
Forward Reaction ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Phosphate Synthase

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0×10-5m and 1.9×102m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3×10-5m; formaldehyde, 4.9×10-4m; d-arabino-3-hexulose 6-phosphate, 7.5×10-5m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0×10-4m; d-fructose 6-phosphate, 1.1×10-3m.

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

1978 ◽  
Vol 24 (9) ◽  
pp. 1017-1023 ◽  
Author(s):  
Hiroshi Onishi ◽  
Osamu Hidaka
Keyword(s):  
Gel Electrophoresis ◽  
Enzyme Production ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Soluble Starch ◽  
Moderately Halophilic ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Acinetobacter Sp ◽  
Sea Sands

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2 M NaCl or 1 M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 °C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.

scholarly journals Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula

1972 ◽  
Vol 128 (5) ◽  
pp. 1241-1253 ◽  
Author(s):  
B. Simonarson ◽  
D. C. Watts
Keyword(s):  
Creatine Kinase ◽  
Specific Activity ◽  
Thiol Group ◽  
Effect Of Temperature ◽  
Thiol Groups ◽  
High Concentration ◽  
Nitrobenzoic Acid ◽  
Purification And Properties ◽  
Michaelis Constants

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl− restores protection to the original value. The quaternary MgADP–creatine–enzyme–nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on Vmax. and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

scholarly journals Partial purification and properties of the two common inherited forms of human erythrocyte adenylate kinase

1972 ◽  
Vol 130 (3) ◽  
pp. 797-803 ◽  
Author(s):  
C. Brownson ◽  
N. Spencer
Keyword(s):  
Adenylate Kinase ◽  
Effect Of Temperature ◽  
Partial Purification ◽  
Heat Denaturation ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Increasing Temperature

1. The partial purification of adenylate kinase, types 1 and 2, from human erythrocytes is described. 2. Gel chromatography of both forms of the enzyme gave estimates of the molecular weights in the range 20000–23000. 3. Studies on crude haemolysates at various pH values indicated that the type 2 enzyme was less stable than the type 1. Heat denaturation studies on the partially purified enzymes confirmed these findings. 4. Measurements of rates of inhibition by iodoacetate and iodoacetamide showed that the type 2 enzyme reacts more readily than the type 1 enzyme with both reagents. 5. The effect of temperature on the initial velocity of ADP formation was measured at a single concentration of both AMP and MgATP2-. The two forms of the enzyme responded differently to increasing temperature.

scholarly journals Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 305
Author(s):  
Suxiao Yang ◽  
Zhemin Liu ◽  
Xiaodan Fu ◽  
Changliang Zhu ◽  
Qing Kong ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Degradation Products ◽  
Alginate Lyase ◽  
Maximal Activity ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
Industrial Preparation ◽  
Alginate Oligosaccharides ◽  
Elimination Mechanism ◽  
G Ratio

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

THE PYRUVIC PHOSPHOFERASE OF ERYTHROCYTES: I. PROPERTIES OF THE ENZYME AND ITS ACTIVITY IN ERYTHROCYTES OF VARIOUS SPECIES

1955 ◽  
Vol 33 (1) ◽  
pp. 38-45 ◽  
Author(s):  
P. F. Solvonuk ◽  
H. R. Collier
Keyword(s):  
Mammalian Species ◽  
Thiol Groups ◽  
Newborn Rats ◽  
Free Thiol ◽  
The Mean ◽  
Very High

Mammalian erythrocytes contain a pyruvic phosphoferase (PPFase) which is activated by K+ and Mg++ and inhibited by Na+ and Ca++. The K+ can be replaced by Rb+ or NH4+, and the Mg++ can be partially replaced by Mn++ or Co++ as activators of the enzyme. The PPFase apparently requires free thiol groups for its activity, as it is completely inhibited by 10−4 M p-chloromercuribenzoate and this inhibition is partially reversed by glutathione. The mean PPFase of the erythrocytes of six mammalian species was determined and found to be in the following order of decreasing activity: man, rat, dog, rabbit, cat, ox. The erythrocytes of chicks and of newborn rats showed a very high PPFase activity.

scholarly journals Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups

1990 ◽  
Vol 272 (2) ◽  
pp. 359-367 ◽  
Author(s):  
J Rúa ◽  
D de Arriaga ◽  
F Busto ◽  
J Soler
Keyword(s):  
Enzyme Inhibitor ◽  
Isocitrate Lyase ◽  
Equilibrium Constants ◽  
Spectrophotometric Titration ◽  
Kinetic Mechanism ◽  
Thiol Groups ◽  
Phycomyces Blakesleeanus ◽  
Denatured State ◽  
Nitrobenzoic Acid ◽  
Dead End

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5′-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.

scholarly journals Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

1998 ◽  
Vol 64 (11) ◽  
pp. 4452-4459 ◽  
Author(s):  
Daniel A. Kunz ◽  
Jui-Lin Chen ◽  
Guangliang Pan
Keyword(s):  
Pseudomonas Fluorescens ◽  
Culture Fluid ◽  
Maximal Activity ◽  
Resonance Spectroscopy ◽  
Keto Acid ◽  
Growth Substrate ◽  
Reaction Products ◽  
Cell Extracts ◽  
Keto Acids ◽  
Essential Components

ABSTRACT Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min−1 ml of culture fluid−1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.

scholarly journals VIII. The combining volumes of hydrogen and oxygen

1916 ◽  
Vol 216 (538-548) ◽  
pp. 393-427 ◽  
Keyword(s):  
Chemical Method ◽  
Fundamental Constant ◽  
Atomic Weight ◽  
Arithmetic Mean ◽  
High Order ◽  
Molecular Weights ◽  
Physico Chemical ◽  
The Subject ◽  
The Individual

The measurement of the combining weights of hydrogen and oxygen has been the subject of so many researches of a high order of excellence that any fresh investigation of this fundamental constant must be submitted with considerable diffidence. Nevertheless, it must be noted that the results obtained by various observers differ appreciably. According to Clarke ( 1 ), the values obtained by Morley and Noyes, by reason of the accuracy of their methods and the close concordance of the individual determinations, outweigh the results of all other investigators. The atomic weight of oxygen being 16, that of hydrogen, according to Morley( 2 ), is 1˙00762, and according to Noyes ( 3 ), 1˙00787. (Clarke, on Noyes’ data, prefers the value 1˙00783.) It is, further, a significant fact that the arithmetic mean of all determinations discussed by Clarke, lies between these two values, which differ by 1 part in 4000. Both values are based on the gravimetric synthesis of water and are independent of a knowledge of the densities of the gases. A physico-chemical method of determining the relative molecular weights depends on the knowledge of the ratio of the densities, together with that of the combining volumes.

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