THE WHEAT LEAF PHOSPHATASES: V. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING β-GLYCEROPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (1) ◽  
pp. 113-120 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
Enzyme Concentration ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Excess Substrate

Wheat leaf press juice contains an enzyme that has β-glycerophosphatase activity, which has a pH optimum close to pH 5.7. The enzyme is inhibited by orthophosphate, pyrophosphate, and 10−4 M fluoride. Fluoride inhibition can be abolished by thorough dialysis. Fluoride partially protects the enzyme from denaturation by heat.Enzyme kinetics shows that the log of the enzyme concentration is proportional to the log of the rate of liberation of orthophosphate from the substrate in the presence of excess substrate (0.2 M to 0.6 M) at pH 5.7. This observation can be used for quantitation of the enzyme.

THE WHEAT LEAF PHOSPHATASES: V. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING β-GLYCEROPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (1) ◽  
pp. 113-120 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
Enzyme Concentration ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Excess Substrate

Wheat leaf press juice contains an enzyme that has β-glycerophosphatase activity, which has a pH optimum close to pH 5.7. The enzyme is inhibited by orthophosphate, pyrophosphate, and 10−4 M fluoride. Fluoride inhibition can be abolished by thorough dialysis. Fluoride partially protects the enzyme from denaturation by heat.Enzyme kinetics shows that the log of the enzyme concentration is proportional to the log of the rate of liberation of orthophosphate from the substrate in the presence of excess substrate (0.2 M to 0.6 M) at pH 5.7. This observation can be used for quantitation of the enzyme.

scholarly journals Degredation of insulin by a particulate fraction from adipose tissue

1970 ◽  
Vol 116 (5) ◽  
pp. 825-831 ◽  
Author(s):  
Kenneth Sumner ◽  
Richard J. Doisy
Keyword(s):  
Adipose Tissue ◽  
Paper Chromatography ◽  
Trichloroacetic Acid ◽  
Degradation Products ◽  
Enzyme System ◽  
Enzyme Concentration ◽  
Particulate Fraction ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Rat Adipose Tissue

The destruction of 125I-labelled insulin by an enzyme system from rat adipose tissue was studied. The system was located in the particulate fraction. Activity was assayed by the amount of 125I-labelled degradation products rendered soluble in trichloroacetic acid. The system was heat-labile, with an alkaline pH optimum. The velocity of the reaction varied directly with the enzyme concentration. Paper chromatography of the degradation products showed six ninhydrin-sensitive areas with radioactivity coinciding with three of them. Albumin inhibited the system; ribonuclease did not. Although only 25% of the total 125I-label was detected by this assay, results with insulin-specific assays suggested that most (80–90%) of the hormone was inactivated. Possible interpretations of these results are discussed. The particulate fractions of other tissues were also studied.

THE WHEAT LEAF PHOSPHATASES: VI. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING ADENOSINE-5′-PHOSPHATE AND PHENOLPHTHALEIN DIPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (5) ◽  
pp. 1275-1281 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
The Other ◽  
Enzymic Hydrolysis ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Hydrolysis Of ◽  
Leaf Juice

At least two enzymes are probably involved in the hydrolysis of mixtures of β-glycerophosphate, phenolphthalein diphosphate, and adenosine-5′-phosphate. One enzyme is primarily responsible for the hydrolysis of β-glycerophosphate whereas the other enzyme hydrolyzes adenosine-5′-phosphate and phenolphthalein diphosphate but has little activity on β-glycerophosphate.The liberation of orthophosphate from adenosine-5′-phosphate and phenolphthalein diphosphate by the enzyme in wheat leaf juice is inhibited by 0.005 M adenosine but not by 0.02 M phosphate. The inhibition of this enzyme by fluoride is markedly smaller than the inhibition of β-glycerophosphatase. The enzyme that hydrolyzes phenolphthalein diphosphate transfers phosphate from phenolphthalein diphosphate to adenosine to form adenosine-5′-phosphate.Experiments on the pH optimum for the enzymic hydrolysis of both adenosine-5′-phosphate and phenolphthalein diphosphate by undialyzed and dialyzed juice preparations with or without added Mg++ suggest that there may be more than one enzyme with different pH optima acting on both adenosine-5′-phosphate and phenolphthalein diphosphate.

Extracellular neuraminidase of Streptomyces albus

1972 ◽  
Vol 18 (7) ◽  
pp. 1007-1014 ◽  
Author(s):  
Marjory K. Myhill ◽  
Thomas M. Cook
Keyword(s):  
Gel Filtration ◽  
Thiobarbituric Acid ◽  
Enzyme Concentration ◽  
Divalent Metal Ions ◽  
Ph Optimum ◽  
Reactive Material ◽  
Culture Filtrates ◽  
Streptomyces Albus ◽  
Excess Substrate ◽  
Fetal Bovine

Extracellular neuraminidase activity (mucopolysaccharide N-acetylneuraminylhydrolase, EC.3.2.1.18) was detected in culture filtrates of Streptomyces albus MA-390, S. albus NCTC 7807, and S. albidoflaviis, but not S. albus ATCC 3004, S. roseochromogenes ATCC 13400, or S. venezuelae ATCC 10595. Neuraminidase of S. albus MA-390, purified 430-fold from crude culture filtrates, cleaved N-acetylneuraminyllactose and released thiobarbituric acid-reactive material from various glycoproteins. With excess substrate the reaction rate was proportional to enzyme concentration. The enzyme was not stimulated by divalent metal ions and was not inhibited by ethylenediaminetetraacetate. Streptomyces albus MA-390 neuraminidase was heat labile and activity was lost rapidly at temperatures of 40°C and above. With N-acetylneuraminyllactose as substrate optimal activity occurred at pH 6.0, but with fetal bovine serum the pH optimum was 3.5. The molecular weight of the enzyme was estimated by gel filtration to be in the range of 40 000 to 45 000.

THE WHEAT LEAF PHOSPHATASES: VI. SOME PROPERTIES OF THE ENZYME SYSTEM HYDROLYZING ADENOSINE-5′-PHOSPHATE AND PHENOLPHTHALEIN DIPHOSPHATE IN CRUDE JUICE PREPARATIONS

1963 ◽  
Vol 41 (1) ◽  
pp. 1275-1281 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Enzyme System ◽  
The Other ◽  
Enzymic Hydrolysis ◽  
Ph Optimum ◽  
Wheat Leaf ◽  
Hydrolysis Of ◽  
Leaf Juice

At least two enzymes are probably involved in the hydrolysis of mixtures of β-glycerophosphate, phenolphthalein diphosphate, and adenosine-5′-phosphate. One enzyme is primarily responsible for the hydrolysis of β-glycerophosphate whereas the other enzyme hydrolyzes adenosine-5′-phosphate and phenolphthalein diphosphate but has little activity on β-glycerophosphate.The liberation of orthophosphate from adenosine-5′-phosphate and phenolphthalein diphosphate by the enzyme in wheat leaf juice is inhibited by 0.005 M adenosine but not by 0.02 M phosphate. The inhibition of this enzyme by fluoride is markedly smaller than the inhibition of β-glycerophosphatase. The enzyme that hydrolyzes phenolphthalein diphosphate transfers phosphate from phenolphthalein diphosphate to adenosine to form adenosine-5′-phosphate.Experiments on the pH optimum for the enzymic hydrolysis of both adenosine-5′-phosphate and phenolphthalein diphosphate by undialyzed and dialyzed juice preparations with or without added Mg++ suggest that there may be more than one enzyme with different pH optima acting on both adenosine-5′-phosphate and phenolphthalein diphosphate.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

THEORY OF THE TRANSIENT PHASE IN AN ENZYME SYSTEM INVOLVING TWO ENZYME-SUBSTRATE COMPLEXES: THE CASE OF THE FORMATION OF PRODUCTS FROM THE FIRST COMPLEX

1959 ◽  
Vol 37 (4) ◽  
pp. 737-743 ◽  
Author(s):  
Ludovic Ouellet ◽  
James A. Stewart
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Active Site ◽  
Substrate Concentration ◽  
Enzyme System ◽  
Kinetic Scheme ◽  
Enzyme Concentration ◽  
Theoretical Treatment ◽  
Transient Phase ◽  
Enzyme Substrate

A theoretical treatment is worked out for the kinetic scheme[Formula: see text]in which the concentration of P1 is followed. The steady-state and transient phase equations are obtained subject to the condition that the substrate concentration is greatly in excess of the enzyme concentration. The conditions under which evidence in favor of this mechanism can be obtained from experimental data are discussed. Under certain conditions, the weight of the enzyme corresponding to one active site can be determined. Methods for the evaluation of the different constants are described.

scholarly journals Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

2012 ◽  
Vol 78 (17) ◽  
pp. 6161-6171 ◽  
Author(s):  
Christoph Sygmund ◽  
Daniel Kracher ◽  
Stefan Scheiblbrandner ◽  
Kawah Zahma ◽  
Alfons K. G. Felice ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Enzyme System ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Carbohydrate Binding ◽  
Ph Optimum ◽  
Content Type ◽  
Polysaccharide Monooxygenase

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

1962 ◽  
Vol 40 (2) ◽  
pp. 165-175 ◽  
Author(s):  
G. C. Becking ◽  
R. O. Hurst
Keyword(s):  
Enzyme Activity ◽  
Ionic Strength ◽  
Enzyme Preparation ◽  
Deoxyribonucleic Acid ◽  
Paper Electrophoresis ◽  
Relative Activity ◽  
Enzyme Concentration ◽  
Uranyl Acetate ◽  
Ph Optimum

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.

Formation of radioactive diacylglycerol from radioisotope-labelled phosphatidylethanolamine by Escherichia coli extracts

1989 ◽  
Vol 67 (6) ◽  
pp. 288-292 ◽  
Author(s):  
H. Aubry ◽  
P. Proulx
Keyword(s):  
Escherichia Coli ◽  
Phospholipase C ◽  
Substrate Concentration ◽  
Incubation Medium ◽  
Isotope Ratio ◽  
Enzyme Concentration ◽  
Water Soluble ◽  
Ph Optimum ◽  
Direct Incorporation

Radioisotope-labelled phosphatidylethanolamine can be converted to radioactive diacylglycerol in the presence of added unlabelled diacylglycerol. With [14C-glycerol; 3H-acyl]phosphatidylethanolamine as substrate, the conversion to double-labelled diacylglycerol occurred without change in isotope ratio indicating that the whole diacylglycerol moiety of phosphatidylethanolamine was directly involved. With [3H-acyl; 32P]phosphatidylethanolamine, formation of [3H]diacylglycerol occurred without production of labelled water-soluble products and consequently no phospholipase C activity could be detected. Under similar conditions, a conversion of [14C-acyl]- or [3H-acyl]-diacylglycerol to labelled phosphatidylethanolamine could also be shown even in the presence of hydroxylamine. [14C-Glycerol; 3H-acyl] diacylglycerol was converted to double-labelled product without change in isotope ratio which again indicated a direct incorporation of the entire diacylglycerol molecule into phosphatidylethanolamine. Both types of conversions were dependent upon time, enzyme concentration, and substrate concentration, and both displayed a pH optimum of approximately 6 and required no added cofactors. In the presence of increasing concentrations of [14C-acyl]diacylglycerol, added to incubation medium containing [3H-acyl]phosphatidylethanolamine, equal amounts of [14C]phosphatidylethanolamine and [3H]diacylglycerol were formed which matched the decrease in [3H]phosphatidylethanolamine. From these results, we conclude that Escherichia coli has an enzyme that catalyses the exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol, with complete sparing of the phosphoethanolamine moiety.Key words: diacylglycerol, phosphatidylethanolamine, exchange, Escherichia coli.

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