TESTOSTERONE METABOLISM IN SUBCELLULAR FRACTIONS OF RAT PROSTATE

1973 ◽  
Vol 73 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Kaoru Nozu ◽  
Bun-ichi Tamaoki
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Inhibitory Effect ◽  
Subcellular Fractions ◽  
Hydrogenase Activity ◽  
Nuclear Fraction ◽  
Cytosol Fraction ◽  
Testosterone Metabolism ◽  
Major Activity

ABSTRACT Homogenates of rat ventral prostates were fractionated into the nuclear (purified from the precipitate at 800× g), mitochondrial (precipitate at 1000–6000 ×g), microsomal (precipitate at 10 000–105 000× g) and cytosol (supernatant fluid at 105 000 × g) fractions, which were morphologically identified under an electron-microscope. Among the subcellular fractions, the highest specific activity of Δ4-5α-hydrogenase was localized in the nuclear and microsomal fractions, while 3α-hydroxysteroid dehydrogenase (E. C. 1. 1. 1.50) activity was concentrated exclusively in the cytosol fraction. By addition of the cytosol fraction, the Δ4-5α-hydrogenase activity in the microsomal fraction was markedly reduced, whereas the activity in the nuclear fraction was scarcely inhibited. By heating the cytosol fraction at 100°C for 20 min, its inhibitory effect was significantly diminished. The inhibitory principle in the cytosol fraction against the Δ4-5α-hydrogenase was mostly concentrated in the precipitate at 0–60% saturation of ammonium sulphate, while the major activity of the 3α-hydroxysteroid dehydrogenase was localized in the precipitate at 40–100% saturation of the salt. According to the enzyme kinetics, the cytosol 0–40 % fraction showed the competitive type of inhibition with the Δ4-5α-hydrogenase activity in the microsomal fraction for testosterone.

scholarly journals Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases

1988 ◽  
Vol 250 (3) ◽  
pp. 713-718 ◽  
Author(s):  
M Söderström ◽  
S Hammarström ◽  
B Mannervik
Keyword(s):  
Microsomal Fraction ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Glutathione Transferases ◽  
Leukotriene C4 ◽  
Cytosol Fraction ◽  
Mastocytoma Cells ◽  
Specific Activities ◽  
Leukotriene A4 ◽  
Microsomal Glutathione

Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4:glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4.

scholarly journals Effect of oestradiol-17β and progesterone on the metabolism of [1,2-3H] progesterone by the rabbit endometrium and myometrium

1976 ◽  
Vol 156 (1) ◽  
pp. 55-62 ◽  
Author(s):  
U Verma ◽  
K R Laumas
Keyword(s):  
Metabolic Pathways ◽  
Hydroxysteroid Dehydrogenase ◽  
Particulate Fraction ◽  
Unit Weight ◽  
Nuclear Fraction ◽  
Uterine Tissue ◽  
Cytosol Fraction ◽  
Control Study

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.

Diacylglycerol acyltransferase in maturing sunflower seeds

2000 ◽  
Vol 28 (6) ◽  
pp. 689-692 ◽  
Author(s):  
S. Triki ◽  
J. Ben Hamida ◽  
P. Mazliak
Keyword(s):  
Enzyme Inhibition ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Subcellular Fractions ◽  
Tween 20 ◽  
Differential Centrifugation ◽  
Sunflower Seeds ◽  
Triton X

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-14C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent Km was 60 μM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

scholarly journals The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

1977 ◽  
Vol 166 (3) ◽  
pp. 429-435 ◽  
Author(s):  
G. Subba Rao ◽  
Leonard N. Norcia ◽  
Joanne Pieringer ◽  
Ronald A. Pieringer
Keyword(s):  
Enzyme Activity ◽  
Rat Brain ◽  
Enzyme Preparation ◽  
Microsomal Fraction ◽  
Inhibitory Effect ◽  
Subcellular Fractions ◽  
Triton X ◽  
Derivatives Of

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3′-phosphate 5′-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3′-phosphate 5′-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg2+ strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent Km for galactosyldiacylglycerol is 200μm, and that for cerebroside is 45μm. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.

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.

STUDIES ON 17β-HYDROXYSTEROID DEHYDROGENASE IN HUMAN ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

1975 ◽  
Vol 79 (1) ◽  
pp. 134-145 ◽  
Author(s):  
K. Pollow ◽  
H. Lübbert ◽  
E. Boquoi ◽  
G. Kreutzer ◽  
R. Jeske ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Undifferentiated Carcinoma ◽  
Subcellular Fractions ◽  
Neoplastic Tissue ◽  
Normal Endometrium ◽  
Proliferative Endometrium ◽  
Well Differentiated ◽  
Degree Of Differentiation

ABSTRACT Specific activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) was measured in subcellular fractions of normal endometrium at different phases of the menstrual cycle, and of endometrial carcinoma at different degrees of differentiation. The purity of fractions was determined by marker enzymes, RNA/DNA ratio or electronmicrographs. Both in normal and neoplastic tissue 17β-HSD activity was located mainly in mitochondria and microsomes. The microsomal enzyme is bound tightly to the membranes of the endoplasmic reticulum. While in normal endometrium specific enzyme activity in subcellular fractions depended on the phase of the cycle, in endometrial carcinoma it depended on the degree of differentiation of the tumours. The highest values of 17β-HSD activity were found in mitochondria and microsomes of early secretory endometrium (factor 10 as compared to proliferative endometrium) and in particulate fractions of well differentiated carcinoma (factor 10 to ⪢ 10 as compared to undifferentiated carcinoma).

ACTIVATION OF SOLUBILIZED STEROID-TRANSFORMING ENZYMES OF ADRENAL MICROSOMAL ORIGIN BY SERUM PROTEINS

1972 ◽  
Vol 54 (2) ◽  
pp. 297-315 ◽  
Author(s):  
MARGOT A. HAMILTON ◽  
R. W. McCUNE ◽  
SIDNEY ROBERTS
Keyword(s):  
High Speed ◽  
Microsomal Fraction ◽  
Adrenal Glands ◽  
Serum Proteins ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fraction ◽  
Rat Serum ◽  
Microsomal Preparation ◽  
Stimulation Of

SUMMARY The reactions which result in the conversion of pregnenolone to progesterone and of progesterone to deoxycorticosterone in undisrupted microsomal preparations from rat adrenal glands were stimulated by homologous serum. The active materials were shown to be firmly associated with serum proteins. The dialysable fraction of serum was either without effect on these transformations or was inhibitory. The enzyme systems involved were partially solubilized by exposure of the microsomal preparation to prolonged sonic treatment or to 1% Triton N-101. After either treatment, 35–40% of the original specific activity of the steroid 21-hydroxylase system responsible for the conversion of progesterone to deoxycorticosterone was found in the supernatant fraction after high-speed centrifugation. However, this solubilized system did not respond to serum preparations. The same procedures also resulted in a supernatant fluid which showed about 50–60% of the initial specific activity of the multi-enzyme system involved in the conversion of pregnenolone to progesterone. In these instances, the stimulatory effect of serum was retained or accentuated. Acetone powders prepared from the adrenal microsomal fraction were also active in the conversion of pregnenolone to progesterone and responded to serum with enhanced activity. Earlier observations that activation of steroid 21-hydroxylase by homologous rat serum was specific for the β-globulin fraction were confirmed in the present investigations. In contrast, stimulation of the conversion of pregnenolone to progesterone was apparently due principally to the albumin fraction. Albumin preparations from a number of other sources, as well as whole human serum protein, were also effective in this regard. The active protein preparations selectively stimulated 4-ene-3β-hydroxysteroid dehydrogenase activity, but did not activate 5-ene-3-oxosteroid isomerase in the microsomal fraction. This finding suggested that activation of 5-ene-3β-hydroxysteroid dehydrogenase was responsible for stimulation of progesterone synthesis from pregnenolone. The results indicate that the protein-bound factor in rat serum which was capable of stimulating the conversion of progesterone to deoxycorticosterone in microsomal preparations from rat adrenal glands was different from that which activates the conversion of pregnenolone to progesterone. Moreover, these diverse factors appeared to act by different mechanisms.

IN VITRO EFFECT OF 16α-HYDROXYPROGESTERONE ON THE ENZYME ACTIVITIES RELATED TO ANDROGEN PRODUCTION IN HUMAN TESTES

1978 ◽  
Vol 88 (4) ◽  
pp. 768-777 ◽  
Author(s):  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Enzyme Activities ◽  
Incubation Medium ◽  
Prostatic Carcinoma ◽  
Microsomal Fraction ◽  
Hydroxysteroid Dehydrogenase ◽  
Steroid Metabolism ◽  
Side Chain ◽  
Cytosol Fraction ◽  
Vitro Effect

ABSTRACT Progesterone was converted in vitro to 16α- and 17α-hydroxyprogesterones in the presence of NADPH by the testicular microsomal fraction (precipitate at 10 000 × g−105 000 × g) obtained from patients with prostatic carcinoma. 16α-Hydroxyprogesterone was not metabolized by either the microsomal or the cytosol fractions, and accumulated in the incubation medium. 16α-Hydroxyprogesterone competitively inhibited the activity of the C-17−C-20 lyase in the testicular microsomal fraction with an estimated inhibitor constant of 72 μm. Moreover, the 16α-hydroxyprogesterone non-competitively inhibited the activity of the 20α-hydroxysteroid dehydrogenase in the testicular cytosol fraction and had an estimated inhibitor constant of 52.9 μm. Other testicular enzymes related to steroid metabolism, such as Δ5-3β-hydroxysteroid dehydrogenase coupled with the Δ4-Δ5 isomerase, 16α-hydroxylase, 17α-hydroxylase and 17β-hydroxysteroid dehydrogenase were not influenced in vitro by 16α-hydroxyprogesterone at the concentration of 0.1 mm. From these findings, it is concluded that 16α-hydroxyprogesterone inhibit specifically the cleavage of the side-chain of 17α-hydroxypregnenes in the course of androgen formation from pregnenolone in vitro.

scholarly journals A novel role for WAVE1 in controlling actin network growth rate and architecture

2015 ◽  
Vol 26 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Meredith O. Sweeney ◽  
Agnieszka Collins ◽  
Shae B. Padrick ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Specific Activity ◽  
Inhibitory Effect ◽  
Actin Network ◽  
Inhibitory Effects ◽  
Cellular Functions ◽  
Network Growth ◽  
Assembly Kinetics ◽  
Filament Networks ◽  
First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

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