Studies on Partially Purified Pig Liver Steroid Δ4-5β-Reductase Activity

1973 ◽  
Vol 51 (12) ◽  
pp. 1661-1668 ◽  
Author(s):  
Edward J. Van Doorn ◽  
John C. Nduaguba ◽  
Albert F. Clark
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Reductase Activity ◽  
Enzymatic Reaction ◽  
Ph Optimum ◽  
Pig Liver ◽  
Liver Cytosol ◽  
End Products ◽  
Tris Maleate

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

scholarly journals Pre-fermentative treatment of a model wine with aim to serve as a functional food with decreased alcohol content

2017 ◽  
Vol 63 (01) ◽  
pp. 47-53
Author(s):  
Irina Mladenoska ◽  
Verica Petkova ◽  
Tatjana Kadifkova Panovska
Keyword(s):  
Enzyme Activity ◽  
Gluconic Acid ◽  
Functional Food ◽  
Ph Value ◽  
Enzymatic Reaction ◽  
Alginate Beads ◽  
High Concentration ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Initial Ph

The effect of substrate concentration on the enzyme activity in the reaction of glucose conversion into gluconic acid was investigated by using three different enzyme preparations in media with two different glucose concentrations. The media were simulating the conditions in the must, thus named as minimal model must, and were composed form combination of several organic acids and glucose. Those media were having initial pH of 3.5 that is a very unfavorable for glucose oxidase activity having a pH optimum at the pH value of 5.5. Among the three preparations used, the bakery additive, Alphamalt Gloxy 5080, was the most active in the medium with glucose concentration of 10 g/L, showing conversion of more than 70% for the period of 24 h, while the same enzyme preparation in the medium with 100 g/L glucose converted only about 7% of glucose. The pH value of the medium at the beginning and at the end of the enzymatic reaction was a good indicator of the enzyme activity. It seems that for the conversion of glucose in higher concentration, enzymatic preparation in high concentration should also be used. The preliminary attempt of immobilization of two preparations of glucose oxidases in alginate beads was also performed and a successful immobilization procedure for utilization in food industry was preliminarily developed. Keywords: glucose oxidases, enzymatic pretreatment, glucose, gluconic acid, model wine, functional food

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.

Biosynthesis Of Gallotannins. ß-Glucogallin-Dependent Galloylation Of 1,6-Digailoylglucose To 1,2,6-Trigalloylglucose

1991 ◽  
Vol 46 (5-6) ◽  
pp. 389-394 ◽  
Author(s):  
Georg G. Gross ◽  
Klaus Denzel
Keyword(s):  
Molecular Weight ◽  
Enzymatic Reaction ◽  
Relative Activity ◽  
The Other ◽  
Ph Optimum ◽  
Acceptor Substrate ◽  
Free Glucose ◽  
Rhus Typhina ◽  
Acyl Donors ◽  
Stimulation Of

An enzyme from leaves of sumach (Rhus typhina) was partially purified that catalyzes the β-glucogallin (l-O-galloylglucose)-dependent galloylation of 1,6-digalloylglucose, thus forming 1,2,6-trigalloylglucose and free glucose. This acyltransferase had a molecular weight of ca. 750,000 and a pH optimum at 5.0-5 .5. Besides β-glucogallin (Km = 3.9 mM ) , also related 1-O-phenylcarboxylglucoses acted as acyl donors. On the other hand, the acceptor substrate, 1,6-digalloylglucose (Km = 0.9 mM ) , could only be replaced by 1,6-diprotocatechuoylglucose (relative activity 46%); however, also tri-, tetra-, and pentagalloylglucoses were galloylated. A pronounced stimulation of the enzymatic reaction was observed upon addition of penta- or hexagalloylglucose into the assay mixtures. The systematic name “ β-glucogallin: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1. - ) is proposed for the enzyme

Steroid Δ4-Reductases of Pig Liver: Partial Purification of Testosterone 5β-Reductase

1971 ◽  
Vol 49 (3) ◽  
pp. 385-392 ◽  
Author(s):  
J. C. Nduaguba ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Soluble Fraction ◽  
Reductase Activity ◽  
Maximum Velocity ◽  
Supernatant Fraction ◽  
Partial Purification ◽  
Pig Liver ◽  
Total Enzyme Activity ◽  
Distribution Studies ◽  
The Stability

Intracellular distribution studies of steroid Δ4-reductase activity in female pig liver were done using testosterone as substrate. About 60% of the total enzyme activity was found in the 100 000 × g soluble fraction. Labelled 17β-hydroxy-5β-androstan-3-one but not 17β-hydroxy-5α-androstan-3-one was isolated from the incubation of 1,2-3H-testosterone with the 100 000 × g supernatant fraction, indicating the presence of 5β-reductase activity. 5β-Reduction may play an important role in the inactivation of some Δ4-3-ketosteroids in the pig liver.Evidence that 5β-reductases differing in substrate specificity are present in the soluble fraction includes (a) variation in the ratios of enzyme activities for several Δ4-3-ketosteroids in different (NH4)2SO4 fractions obtained from the 100 000 × g soluble fraction, (b) kinetic data showing that the maximum velocity for an equimolar mixture of testosterone and hydrocortisone is the sum of the maximum velocities for the substrates when used singly, and (c) separation of the enzyme activity specific for testosterone from that specific for hydrocortisone by use of Sephadex G-100 and hydroxylapatite chromatography.Utilizing (NH4)2SO4 precipitation and chromatography on Sephadex G-100 and hydroxylapatite, a 105-fold purification of testosterone Δ4-5β-reductase from the 100 000 × g supernatant fraction has been attained. The presence of 5 mM β-mercaptoethanolamine increased the stability of the enzyme.

Properties and characteristics of partly purified glutamate dehydrogenase from sheep rumen mucosa

1981 ◽  
Vol 46 (11) ◽  
pp. 2766-2773
Author(s):  
Katarína Holovská ◽  
Viera Lenártová ◽  
Ivan Havassy
Keyword(s):  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymatic Reaction ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ph Optimum ◽  
Relative Molecular Weight ◽  
Sheep Rumen ◽  
The Individual

The purification of glutamate dehydrogenase from sheep rumen mucosa on DEAE-cellulose afforded two enzyme fractions with glutamate dehydrogenase activity. The enzyme fraction II (tissue glutamate dehydrogenase) was freed of contaminating proteins in the subsequent purification step on Sephadex G-200. The approximate relative molecular weight (260 000) of tissue glutamate dehydrogenase (fraction II) was determined by gel filtration on Sephadex G-200 and the approximate relative molecular weight of its polypeptide chain (48 000) was established by polyacrylamide gel electrophoresis in SDS. The pH-optimum of fraction II was 7.9. The effect of substrate concentration on the rate of the enzymatic reaction was examined and the following apparent Michaelis' constants were found for the individual substrates: NADH 6.25 . 10-5 mol/l, 2-oxoglutarate 4.5 . 10-3 mol/l, and NH4+ 77 . 10-3 mol/l.

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa

1979 ◽  
Vol 25 (12) ◽  
pp. 1381-1386 ◽  
Author(s):  
Kenji Yamamoto ◽  
Mitsuaki Moriguchi ◽  
Hiroyasu Kawai ◽  
Tatsurokuro Tochikura
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Neurospora Crassa ◽  
Isoelectric Point ◽  
Enzyme Preparation ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Uridine Diphosphate ◽  
Chromatographic Techniques

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4.Maximum activity of the enzyme was observed at pH7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate.The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.

3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Spruce ( Picea abies). Properties and Regulation

1990 ◽  
Vol 45 (9-10) ◽  
pp. 973-979 ◽  
Author(s):  
Meinrad Boll ◽  
Angelika Kardinal
Keyword(s):  
Enzyme Activity ◽  
Picea Abies ◽  
Cell Suspension ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cell Suspension Cultures ◽  
Callus Cultures ◽  
Suspension Cultures ◽  
Phase Cell ◽  
Ph Optimum

Abstract HM GCoA reductase was identified in seedlings, callus cultures, cell suspension cultures and in needles of spruce ( Picea abies) (L.) (Karst). Activity was found in both the 18 K pellet and in the 105 K pellet with different ratios between the two fractions from the various sources. The enzyme has a pH-optimum of 7.9 and an absolute requirement for NADPH . The presence of a thiol reagent such as dithiothreitol is required for activity. Km for HM G CoA is 20 -25 μM. Detergents have differential effects on the activity. In seedlings, enzyme activity was considerably higher in the hypocotyls than in the cotyledons. Enzyme activity was high in dark-grown and low in light-grown seedlings. When the light conditions were reversed, levels of activity adapted to the respective new conditions (increase or decline of specific activity). Aerobic incubations of seedlings, callus cultures or needles in medium containing a carbon source, resulted in a large (up to 20-fold) transient increase of HMGCoA reductase activity. Transfer of stationary phase cell suspension cultures into new medium caused a similarly large increase of activity. A number of carbohydrates induced the enzyme, glucose, fructose and sucrose being most effective. The increase of activity was prevented by cycloheximide. All changes of activity were much more pronounced in the 18 K pellet HMG CoA reductase

scholarly journals Isolation of the liver N-aspartyl-β-glucosaminidase in aspartylglucosaminuria

1976 ◽  
Vol 153 (3) ◽  
pp. 749-750 ◽  
Author(s):  
H Savolainen
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzyme Molecule ◽  
Ph Optimum ◽  
Normal Enzyme

The isolation of liver N-aspartyl-beta-glucosaminidase in human aspartylglucosaminuria, where this enzyme activity is diminished, yields an enzyme molecule with the same molecular weight and pH optimum as the normal enzyme. Its activity is 10% of that of the control preparation. Combination of both enzymes results in the summation of both activities, and the pathological enzyme does not inhibit the control preparation. It is concluded that no change into a totally different isoenzyme has occurred in aspartylglucosaminuria.

Purification and properties of a nonspecific β-fructofuranosidase (inulinase) from the mushroom Panaeolus papillonaceus

1987 ◽  
Vol 33 (6) ◽  
pp. 520-524 ◽  
Author(s):  
Khana Mukherjee ◽  
S. Sengupta
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Activity Ratio ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Inulinase Activity ◽  
Wide Ph Range ◽  
Total Molecular Weight ◽  
Ph Range

A nonspecific β-fructofuranosidase (inulinase) was purified to electrophoretic homogeneity from the culture filtrate of the mushroom Panaeolus papillonaceus. The enzyme is the first purified from a basidiomycete and consists of two subunits with a total molecular weight of 116 000. It is most active on sucrose, then on raffinose, stachyose, and inulin, in decreasing order. The sucrase/inulinase activity ratio (S/I) is 5.7. Fructose was detected as the liberated sugar from raffinose, stachyose, and inulin. The enzyme is highly thermostable with an optimum temperature range of 60–65 °C and a pH optimum of 6.0. The enzyme is stable over the pH range 4–10, and is also active over a wide pH range, exhibiting 50% activity even at pH 8.5. Iodoacetate, azide, and EDTA, at 20 mM concentration, and 1% (w/v) SDS have no effect on enzyme activity, whereas Ag+ and Hg2+ at 2 mM are highly inhibitory.

Production and partial characterization of elastase of Bacillus subtilis isolated from the cervices of human females

1988 ◽  
Vol 34 (7) ◽  
pp. 855-859 ◽  
Author(s):  
Mohinder Kaur ◽  
K. K. Tripathi ◽  
Meenakshi Gupta ◽  
P. K. Jain ◽  
M. R. Bansal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Ammonium Sulphate ◽  
Phosphate Buffer ◽  
Gel Filtration ◽  
Ammonium Sulphate Precipitation ◽  
Tris Maleate ◽  
Culture Supernatants

Conditions are described for the production of extracellular elastase by Bacillus subtilis. The yield of enzyme was maximum in shake–cultures grown in Syncase medium at 37 °C and was stable in culture supernatants. The enzyme, purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration, showed a molecular weight of 25 000 and activity between pH 6.0 and 9.5, with an optimum of 9.0 in Tris–maleate buffer. Elastinolytic activity was maximum in glycine–NaOH buffer and minimum in phosphate buffer. Enzyme activity was adversely affected by temperature ≥ 40 °C.

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