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.

Secretagogue-induced changes in subcellular Ca2+ distribution in isolated pancreatic acini

1981 ◽  
Vol 240 (2) ◽  
pp. G130-G140
Author(s):  
R. L. Dormer ◽  
J. A. Williams
Keyword(s):  
Atomic Absorption Spectrometry ◽  
High Speed ◽  
Microsomal Fraction ◽  
Absorption Spectrometry ◽  
Subcellular Fractionation ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Induced Changes ◽  
Stimulation Of

In a prior study, we demonstrated that pancreatic secretagogues increased both the uptake into and washout of 45Ca2+ from isolated mouse pancreatic acini. The net result of these processes was an initial fall in total acinar cell Ca2+ content. In the present study, we have employed subcellular fractionation of acini under conditions that minimized posthomogenization redistribution of Ca2+ in order to localize those organelles involved in intracellular Ca2+ fluxes. Homogenization and differential centrifugation of acini, preloaded with 45Ca2+ and subjected to a period of washout, showed that carbachol induced an increased loss of 45Ca2+ from all fractions isolated. The high-speed microsomal fraction lost 45Ca2+ to a greater extent than did whole acini; measurement of total Ca2+ by atomic absorption spectrometry showed a net loss of Ca2+ from this fraction. Purification of the lower-speed fractions indicated that carbachol increased 45Ca2+ exchange with both zymogen granules and mitochondria, but net Ca2+ levels in these organelles were unchanged. It was concluded that stimulation of pancreatic acini by carbachol results in the release of calcium from a microsomal compartment leading to a rise in cytoplasmic Ca2+, increased exchange with granule and mitochondrial Ca2+, and increased efflux of Ca2+ from the cell.

scholarly journals Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation

1989 ◽  
Vol 263 (2) ◽  
pp. 565-572 ◽  
Author(s):  
D Riendeau ◽  
D Denis ◽  
L Y Choo ◽  
D J Nathaniel
Keyword(s):  
Lipid Peroxidation ◽  
High Speed ◽  
Chemical Reduction ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Lipoxygenase Activity ◽  
Supernatant Fraction ◽  
Acid Oxidation ◽  
Thiol Compounds ◽  
Stimulation Of

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1974 ◽  
Vol 61 (3) ◽  
pp. 789-807 ◽  
Author(s):  
Gert Kreibich ◽  
David D. Sabatini
Keyword(s):  
Serum Proteins ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Rat Serum ◽  
Coomassie Blue ◽  
Specific Subset ◽  
Low Levels ◽  
Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

scholarly journals BRAIN MITOCHONDRIA

1963 ◽  
Vol 19 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Diana S. Beattie ◽  
Howard R. Sloan ◽  
R. E. Basford
Keyword(s):  
High Speed ◽  
Brain Cortex ◽  
Lactate Production ◽  
Mitochondrial Fraction ◽  
Brain Mitochondria ◽  
Glycolytic Enzymes ◽  
Supernatant Fraction ◽  
Glycolytic Activity ◽  
High Speed Supernatant ◽  
Stimulation Of

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.

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 A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy

1973 ◽  
Vol 134 (1) ◽  
pp. 103-112 ◽  
Author(s):  
E. Heather Mangiapane ◽  
Katherine A. Lloyd-Davies ◽  
David N. Brindley
Keyword(s):  
Partial Hepatectomy ◽  
Microsomal Fraction ◽  
Actinomycin D ◽  
Specific Activity ◽  
Supernatant Fraction ◽  
Glycerol Phosphate ◽  
Liver Remnant ◽  
Control Value ◽  
Phosphatidate Phosphohydrolase ◽  
Subtotal Hepatectomy

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.

scholarly journals The metabolism of phosphatidylinositol in the thyroid gland of the pig

1971 ◽  
Vol 123 (1) ◽  
pp. 19-33 ◽  
Author(s):  
F. B. Jungalwala ◽  
N. Freinkel ◽  
R. M. C. Dawson
Keyword(s):  
Microsomal Fraction ◽  
Water Soluble ◽  
Supernatant Fraction ◽  
Soluble Product ◽  
Phosphorus Containing ◽  
Optimum Ph ◽  
Lipid Precursor ◽  
Specific Stimulation ◽  
Rapid Transfer ◽  
Stimulation Of

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca2+-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3–5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-3H1]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [3H]inositol and 32P, and then incubated with unlabelled inositol, there was a dramatic loss of 3H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of 32P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-3H1]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.

Exchange of iodine-131-labeled chylomicron protein in vitro

1960 ◽  
Vol 199 (3) ◽  
pp. 433-436 ◽  
Author(s):  
Alan F. Hofmann
Keyword(s):  
Serum Protein ◽  
Serum Proteins ◽  
Marked Decrease ◽  
Specific Activity ◽  
Rat Serum ◽  
Before And After ◽  
Normal Rat ◽  
Iodine 131 ◽  
Protein Specific Activity

Chylomicrons isolated from rat chyle were iodinated with iodine131 and the protein specific activity determined before and after incubation with normal rat serum. A marked decrease in chylomicron protein specific activity occurred during incubation because of adsorption of nonradioactive soluble serum protein as well as exchange of part of the labeled chylomicron protein with lipoprotein protein. Ultracentrifugal fractionation of serum after such incubation revealed significant radioactivity in all of the lipoprotein fractions, but with low specific activity reflecting dilution by larger protein pools. Serum proteins with density greater than 1.21 contained much less radioactivity.

THE MECHANISM OF ACTION OF GONADOTROPHIC HORMONES IN AMPHIBIANS: THE STIMULATION OF Δ5-3β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN TESTES OF XENOPUS LAEVIS DAUDIN

1970 ◽  
Vol 47 (4) ◽  
pp. 439-NP ◽  
Author(s):  
J. P. WIEBE
Keyword(s):  
Xenopus Laevis ◽  
Microsomal Fraction ◽  
Follicle Stimulating Hormone ◽  
Fold Increase ◽  
Hydroxysteroid Dehydrogenase ◽  
Human Chorionic Gonadotrophin ◽  
Spectrophotometric Methods ◽  
Pregnant Mare Serum Gonadotrophin ◽  
Stimulation Of

SUMMARY The effects of gonadotrophins on the Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) activity in testes of Xenopus laevis have been determined by histochemical and spectrophotometric methods. Methallibure was employed to inhibit the activity of endogenous pituitary gonadotrophins. Injections of pregnant mare serum gonadotrophin (PMS), human chorionic gonadotrophin (HCG), follicle stimulating hormone (FSH), or luteinizing hormone (LH) resulted in pronounced increases in 3β-HSD activity in the microsomal fraction. PMS was the most stimulatory; the injection of 250 i.u. resulted in an almost ten fold increase in activity. PMS and HCG were more effective than FSH and LH in stimulating 3β-HSD activity and PMS and FSH were more effective than HCG and LH respectively. The increases in activity were inhibited by concomitant injections of actinomycin. 3β-HSD activity was not stimulated directly by gonadotrophins in vitro, but another dehydrogenase or group of dehydrogenases appeared to be activated directly by HCG, FSH and LH but not by PMS. The results are discussed in relation to findings in mammals.

scholarly journals The In Vivo Uptake and Incorporation of Radioisotopes into Proteins of Wheat Endosperm

1964 ◽  
Vol 17 (1) ◽  
pp. 102 ◽  
Author(s):  
Janet SD Graham ◽  
RK Morton ◽  
JK Raison
Keyword(s):  
High Speed ◽  
Storage Proteins ◽  
Specific Activity ◽  
Soluble Proteins ◽  
Protein Bodies ◽  
Supernatant Fraction ◽  
Pulse Labelling ◽  
Wheat Endosperm ◽  
In Vivo Uptake

A procedure is described for the quantitative i'301ation of the protein bodies from developing wheat endosperm. The changes with time in specific a.ctivity of the storage proteins (present in the protein bodies) and of the soluble proteins (present in the high-speed supernatant fraction) have been followed in "pulse"labelling experiments. Excised wheat heads were exposed to [35SJsulphate for periods of 5 min to 1 hr; dilution of the 35S-radioactivity within the wheat head before the incorporation of radioactivity into the endosperm proteins resulted in a constant ratio of the specific activity of the soluble proteins to that of the storage proteinfl.

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