3α‐Hydroxysteroid: NAD Oxidoreductase Activity in Crystalline Preparations of 20β‐Hydroxysteroid: NAD Oxidoreductase

Pocklington, T; Jeffery, Jonathan

doi:10.1111/j.1432-1033.1968.tb19574.x

Cited by 24 publications

(9 citation statements)

References 15 publications

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“…4,ug/l, in reasonable agreement with values determined by much more laborious methods (Ismail & Harkness, 1967;van der Molen & Corpechot, 1968;Schubert, Wehrberger, Wachtel & Schlegel, 1969). Although the present results clearly establish the practicability of the employed analytical approach, any claim to the intrinsic specificity of the method must be qualified by the finding of 30x-hydroxy steroid oxidoreductase activity in preparations of the enzyme used (Pocklington & Jeffery, 1967).…”

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confidence: 68%

Formation of hydrogen sulphates as a means of separating alcoholic from non-alcoholic steroids

Norymberski¹,

Riondel²

1970

Biochemical Journal

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Sobel, Drekter & Natelson (1936) determined plasma cholesterol by a method that involved the formation of cholesteryl hydrogen sulphate as a means of separating the sterol from its esters and from other non-alcoholic substances. It is now shown that under appropriate conditions the analytical approach of Sobel et al. (1936) is capable of wider application. Each compound tested (see Table 1) was dissolved in dry pyridine (0.2ml), and the solution was chilled in ice-water and mixed with a saturated solution of chlorosulphonic acid in carbon tetrachloride (0.1 ml; a fresh solution was prepared weekly). After 30min at room temperature, 0.4M-NaHCO3 (1.0ml) was added and the non-alcoholic fraction extracted with benzene (2 x 2.5ml) or (in the cases of cortisol and aldosterone) with dichloromethane (2 x 2.5ml). The extract was washed with water (0.5ml) and evaporated to dryness. For solvolysis the aqueous phases were combined and diluted with 2M-pyridinium sulphate (1.0ml) and extracted with dichloromethane (2 x 2.5ml), and the extract was evaporated to dryness; the residue was kept in dioxan (1 ml) for 4h at 370C, and the solution was diluted with dichloromethane (4ml), washed with 0.4M-NaHCO3 (0.5ml) and evaporated to dryness (McKenna & Norymberski, 1960), giving the alcoholic fraction. In the case of pregnanediol the aqueous phase was adjusted to pHl, mixed with ammonium sulphate (0.75g) and extracted with ether-ethanol (3: 1, v/v; 3 x 2ml) (Edwards, Kellie & Wade, 1953); solvolysis was in dioxan containing 2% (v/v) ofethanol at 470C. For enzymic hydrolysis the aqueous phase was adjusted to pH4.9 with acetic acid; 2M-sodium acetate buffer, pH4.9 (0.2ml), was added and then the gastric juice of Helix pomatia (Henry & Thevenet, 1952; supplied by l'Industrie Biologique Fran9aise, Gennevilliers, France) containing 8000 sulphatase units (Roy, 1956). After 48h at 370C the mixture was extracted with dichloromethane (3 x 2ml), and the extract was washed with 0.4M-NaHCO3 (0.5ml) and evaporated to dryness. The minor fraction and part of the major fraction (see Table 1

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confidence: 68%

Formation of hydrogen sulphates as a means of separating alcoholic from non-alcoholic steroids

Norymberski¹,

Riondel²

1970

Biochemical Journal

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“…Early kinetic findings [3] appeared to indicate that these activities depended upon more than one centre, but further work [a] established that the simple assay procedures originally used gave approximate estimates of the kinetic constants which could be considerably in error, so that the question of one or more centres remained open. Assay problems included the low solubility of many steroids in aqueous media, and instability of the enzyme under some conditions.…”

Section: Crystalline Preparations Of Cortisone Reductase An Enzyme Inmentioning

confidence: 99%

Relationships between the 3α‐and 20β‐Hydroxysteroid

Gibb

Jeffery

1971

European Journal of Biochemistry

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Crystalline cortisone reductase prepared from Streptomyces hydrogenans has 3a-and 20p-hydroxysteroid: NAD oxidoreductase activity. The total rate of reaction for mixtures of 17a,21-dihydroxypregn-4-ene-3,l 1,20-trione with 5a-androstan-3,16-dione, and of 1 'Ip-hydroxy-1 amethyl-5n-androstan-3-one with either of these compounds indicated that these substrates do not react independently. 3p-Hydroxy-5a-androstan-16-one inhibited the reduction of both 17a,21-dihydroxypregn-4-ene-3,il ,2O-trione and 5a-androstane-3,l 6-dione.20p-Hydroxypregn-4-en-3-one also inhibited the reduction of both these substrates. The findings allow the view that the 301-and 208-activities may depend upon a single active centre with complex binding characteristics. [a] established that the simple assay procedures originally used gave approximate estimates of the kinetic constants which could be considerably in error, so that the question of one or more centres remained open. Assay problems included the low solubility of many steroids in aqueous media, and instability of the enzyme under some conditions. Kinetic measurements have now been made using certain favourable steroids under conditions which give good reproducibility. Kinetic constants were calculated, and estimates made of the probable errors. These findings show that the 301-and 20g-activities are not independent. The relationship between them is discussed. MATERIALS AND METHODS Steroids GmbH (Mannheim, Germany) and thin layer adsorbants from E. Merck A.G. (Darmstadt, Germany).Enzyme solution sufficient for one experiment was made up immediately before use by diluting stock enzyme suspension (5 mg/ml2.2 mM ammonium sulphate, pH 7.0) with 5 mM Tris containing 1 mg/ml EDTA (disodium salt), pH 8.2, and the enzyme solution was kept a t 4 "C during the course of the experiment. The NADH solution was made up immediately before use in 10 mM Tris, pH 8.2.135 mM Sodium-phosphate buffer, pH 7.0 (2 ml) containing 1.35mM EDTA (disodium salt) and 4.05 mM NADH solution (0.1 ml) were put into a spectrophotometer cell of 1 cm light path, maintained a t 25 "C by a water jacket. Enzyme solution (volume depending upon the amount of enzyme required) and water (to make 0.56 ml) were then added. Dimethyl sulphoxide (0.02 ml) alone, or containing the steroid inhibitor when one was used, was added next. The reaction was initiated by addition of steroid substrate in 0.02 ml dimethyl sulphoxide. For mixed substrate reactions, the solutions of steroid in dimethyl sulphoxide were mixed and reaction initiated by a single addition of 0.04 ml of this solution. Spectrophotometric measurements were made at 340 nm using a Unicam SP-800 double-beam recording spectrophotometer (Unicam Instruments, Cambridge, England). The reference cell contained solutions as above, except that water was added instead of enzyme, dimethyl sulphoxide instead of substrate solution, and 3.24mM NADH instead of 4.05mM NADH. The initial absorbance reading of the reaction cell relative to the reference cell was approximately 0.2 units. The...

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“…In the steroid field, Hurlock & Talalay (1956 used the 3a-hydroxy steroid oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni to assay 3a-ols and 3-ones of the androstane and pregnane series and the 3fi,17fi-hydroxy steroid oxidoreductase (EC 1.1.1.51) from the same source to assay 3f,and 17fi-ols and the corresponding ketones of the androstane series. Hubener's 20fl-hydroxy steroid oxidoreductase (EC 1.1.1.53) from Streptomyces hydrogenans (Hiibener & Lehmann, 1958;Hubeneretal., 1959;Hiibener & Sahrholz, 1960), later shown to catalyse also the interconversion of 3a-ols and 3-ones (Pocklington & Jeffery, 1967; Gibb & Jeffery, 1972, 1973a, has been used to assay 20-oxoand 2011-hydroxypregnanes (Henning & Zander, 1962;Hubener, 1962;Rick, 1962).…”

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confidence: 99%

Radio-enzymic assay of oxo steroids by their reduction with the tritiated form of reduced nicotinamide-adenine dinucleotide

Norymberski¹,

Stoddart²,

Wolstenholme³

1975

Biochemical Journal

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A method of analysis is proposed based on the enzyme-catalysed transfer of tritium from [3H]NADH to suitable substrates. Its practicability is demonstrated on the examples of oestrone and progesterone with the respective use of the 3 beta, 17 beta- and 3 alpha, 20 beta-hydroxysteroid oxidoreductase. Specificity is tested by application to the analysis of the plasma of pregnant women and measurement of the 3H/14C ratios on purification of the enzymic reduction products.

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3α‐Hydroxysteroid: NAD Oxidoreductase Activity in Crystalline Preparations of 20β‐Hydroxysteroid: NAD Oxidoreductase

Cited by 24 publications

References 15 publications

Formation of hydrogen sulphates as a means of separating alcoholic from non-alcoholic steroids

Formation of hydrogen sulphates as a means of separating alcoholic from non-alcoholic steroids

Relationships between the 3α‐and 20β‐Hydroxysteroid

Radio-enzymic assay of oxo steroids by their reduction with the tritiated form of reduced nicotinamide-adenine dinucleotide

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