scholarly journals Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5α-reductase

1973 ◽  
Vol 132 (2) ◽  
pp. 283-291 ◽  
Author(s):  
Paul V. Maynard ◽  
Euan H. D. Cameron
Keyword(s):  
Cytochrome C ◽  
Microsomal Fraction ◽  
Soluble Fraction ◽  
Subcellular Fractions ◽  
Dehydrogenase System ◽  
Separate System ◽  
Hydroxy Steroid ◽  
Microsomal Pellet ◽  
Steroid Dehydrogenase

The location and some characteristics of rat adrenal C19-steroid 5α-reductase were investigated by using [7α-3H]androst-4-ene-3,17-dione and [7α-3H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3α- and 3β-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17β-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C19-steroid 5α-reductase, which showed similar values for Km and Vmax. when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5α-androstane-3,17-dione and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5α-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5α-reduction of C19- and C21-steroids in the rat adrenal.

A metabolic stability determination of Tetrahydrothiazolopyridine derivative a selective 11β-hydroxy steroid dehydrogenase type 1 (11β-hsd1) inhibitor

2017 ◽  
Vol 8 (3) ◽  
Author(s):  
MURUGESH KANDASAMY ◽  
MUHAMMED SALIHIN ◽  
MALLIKARJUNA RAO PICHIKA ◽  
SLAVKO KOMARNYTSKY ◽  
THIRUMURUGAN RATHINASABAPATHY
Keyword(s):  
Metabolic Stability ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

scholarly journals Mechanistic and stereochemical studies on 3-oxo steroid Δ4-Δ5-isomerase from human placenta

1980 ◽  
Vol 185 (2) ◽  
pp. 411-421 ◽  
Author(s):  
M Akhtar ◽  
M Calder ◽  
T Smith ◽  
J N Wright
Keyword(s):  
Double Bond ◽  
Hydrogen Atom ◽  
Human Placenta ◽  
Microsomal Fraction ◽  
Isomerization Reaction ◽  
Acidic Group ◽  
Incoming Proton ◽  
Membrane Bound ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.

Chromogenic Assays for the Determination of Prothrombin Related Material in Rat Liver Fractions.

1979 ◽  
Author(s):  
Linda J Beecroft
Keyword(s):  
Rat Liver ◽  
Vitamin K ◽  
Microsomal Fraction ◽  
Liver Homogenate ◽  
Chromogenic Substrates ◽  
Related Material ◽  
Esterolytic Activity ◽  
Echis Carinatus ◽  
Microsomal Pellet

Clotting assays are not easily applied to turbid solutions such as microsomal fractions. With the development of chromogenic substrates, the esterolytic activity of prothrombin related material can be assayed biochemically in such systems. Liver fractions were prepared by differential centrifugaron. Liver homogenate was centrifuged at 10,000 g. for 10 minutes to yield supernatant 1.Supernatant 1 was further centrifuged at 105,000 g for 60 minutes to yield the microsomal pellet and supernatant 2. Taipan and Echis carinatus snake vanoms were used to generate esterolytic activity in the various liver fractions. In all fractions the esterolytic activity generated by E. carinatus venom was greater than that generated by Taipan Venom. Both assays indicated that the microsomal pellet had similar esterolytic activity to supernatant 2. When liver fractions were prepared from warfarin treated rats, the assays revealed that the relative proportions of prothrombin related material in the fractions had altered. The esterolytic activity of the microsomal fraction was found to be greatly increased whilst supernatant 2 had no detectable activity.Warfarin treated rats have greatly decreased levels of prothrombin in the plasma due to inhibition of vitamin K-dependent carboxylation in the liver. It is suggested that the lack of prothrombin in the plasma reflects the lack of soluble prothrombin in supernatant 2, and the concomitant build-up of precursor forms bound to the microsomes.

scholarly journals 3β-Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates

1971 ◽  
Vol 125 (4) ◽  
pp. 983-989 ◽  
Author(s):  
R S Basch ◽  
M J Finegold
Keyword(s):  
Electron Microscopy ◽  
Microsomal Fraction ◽  
Total Activity ◽  
Mitochondrial Fraction ◽  
Differential Centrifugation ◽  
Radioisotopic Method ◽  
Cell Fractions ◽  
Kinetics Of ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The activity of 3β-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent Km for pregnenolone, dehydroepiandrosterone or NAD+, but demonstrated a distinct difference in the Km for NADP+. pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.

scholarly journals Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification

1967 ◽  
Vol 105 (2) ◽  
pp. 427-442 ◽  
Author(s):  
N. F. González-Cadavid ◽  
P. N. Campbell
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Ammonium Phosphate ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Nuclear Fraction ◽  
Extraction And Purification

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4·0 with distilled water and the other extracted from the residues of the first extraction with 0·15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0·3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate–neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123μg. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24·4; mitochondrial fraction, 57·2; heavy microsomal fraction, 5·2; standard microsomal fraction, 10·6; cell sap, 2·7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate–neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4·0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75–80%.

scholarly journals The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1967 ◽  
Vol 105 (2) ◽  
pp. 443-450 ◽  
Author(s):  
N. F. González-Cadavid ◽  
P. N. Campbell
Keyword(s):  
Sodium Chloride ◽  
Cytochrome C ◽  
Total Protein ◽  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Subcellular Fractions ◽  
Nuclear Fraction ◽  
Mitochondrial Cytochrome ◽  
Peak Value ◽  
Mitochondrial Cytochrome C

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [14C]lysine and killed 7·5, 15, 30 and 60min. later. The livers were homogenized in 0·3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4·0 and then with 0·15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [14C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0·15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0·15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4·0.

SUBCELLULAR LOCALIZATION OF HUMAN PLATELET LIPOXYGENASE ACTIVITY: COMPARISON BETWEEN NORMAL AND LIPOXYGENASE-DEFICIENT PLATELETS

1987 ◽  
Author(s):  
M Okuma ◽  
K Kanaji ◽  
F Ushikubi ◽  
H Uchino
Keyword(s):  
High Performance ◽  
Microsomal Fraction ◽  
Human Platelet ◽  
Normal Subjects ◽  
Reversed Phase ◽  
Lipoxygenase Activity ◽  
Subcellular Fractions ◽  
Platelet Suspension ◽  
Activity Comparison

Lipoxygenase activities were estimated in platelet subcellular fractions as well as in intact platelets obtained from normal subjects and patients with deficient platelet lipoxygenase activities (<mean - 2SD of normal activities). From a washed platelet suspension (intact platelets), subcellular fractions including 12,000 x g supernatant of sonicated platelets (F-I), 105,000 x g supernatant (cytosol, F-II) and sediment (microsomal fraction, F-III) of F-I were prepared by differential centrifugation at 4°C. The enzyme activity was studied by the determination of 12-hy-droxyeicosatetraenoic acid (HETE; ng) produced by the reaction of 100μM arachidonic acid with 108 platelets or the subcellular fraction derived from them at pH 7.4 for 5 min at 37°C in the presence or absence of 2.5 mM CaCl2 and/or 2 mM ATP by the use of reversed-phase high-performance liquid chromatography. In experiments with subcellular fractions, reduced glutathione was added to the reaction mixture. In normal subjects, HETE production by intact platelets, F-I, F-II and F-III was 1,162.4±203.3, 1,029.7±403.8, 368.8±175.8 and 194.4±73.4 (M±SD, n=9), respectively, and was not significantly affected by the addition of CaCl2 and/or ATP. HETE produced by 12,000 x g sediment of sonicated platelets was negligible (<1 % of the product by intact platelets). One of the 7 patients showed no detectable lipoxygenase activity both in intact platelets and in any subcellular fractions, while, the other 6 patients showed reduced lipoxygenase activities in all subcellular fractions as well as in intact platelets: HETE produced by intact platelets, F-I, F-II and F-III was 78.8± 112.3, 59.1± 36.3, 37.0± 18.9 and 17.7±15.8 (n=6), respectively. The addition of CaCl2 significantly increased HETE production only by the patient’s F-I (p< 0.02),while ATP showed no significant effect in any experiments.Thus, it was shown that lipoxygenaseactivities were not fully exhibited in intact platelets with the deficient enzyme activities and that F-I could produce more HETE than intact platelets especially in the presence of CaCl2 only in the case of such patient’s platelets.

scholarly journals Subcellular localization and properties of mouse adrenal C19-steroid 5β-reductase

1975 ◽  
Vol 147 (1) ◽  
pp. 165-174 ◽  
Author(s):  
W Collins ◽  
E H Cameron
Keyword(s):  
Substrate Concentration ◽  
Soluble Fraction ◽  
Product Concentration ◽  
High Substrate Concentration ◽  
Ph Optimum ◽  
Metabolizing Enzymes ◽  
Small Activity ◽  
Substrate Concentrations ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The localization and some characteristics of mouse adrenal C19-steroid 5 β-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 α-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3α-, 3β- and a small amount of 17 β-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 β-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 β products first had to be oxidized to 5 β-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 β-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 β-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 β reduction of [7 α-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) ±0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.

scholarly journals ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER

1974 ◽  
Vol 61 (1) ◽  
pp. 188-200 ◽  
Author(s):  
Henri Beaufay ◽  
Alain Amar-Costesec ◽  
Ernest Feytmans ◽  
Denise Thinès-Sempoux ◽  
Maurice Wibo ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Analytical Study ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome ◽  
Nadph Cytochrome C Reductase

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b5 and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-ß-glucosaminidase, ß-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.

Ab initio structure determination of cytochrome c6 by combined reciprocal space-real space approach

2003 ◽  
Vol 218 (1) ◽  
Author(s):  
K. Chowdhury ◽  
S. Ghosh ◽  
M. Mukherjee
Keyword(s):  
Cytochrome C ◽  
Ab Initio ◽  
Structure Determination ◽  
Direct Method ◽  
Real Space ◽  
Reciprocal Space ◽  
Cytochrome C6 ◽  
Ab Initio Structure ◽  
Space Approach

AbstractThe direct method program SAYTAN has been applied successfully to redetermine the structure of cytochrome c

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