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 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.

Effects of manganese on Q-T interval and distribution of calcium in rat heart

1963 ◽  
Vol 205 (6) ◽  
pp. 1209-1212 ◽  
Author(s):  
Loyal L. Conrad ◽  
Donald J. Baxter
Keyword(s):  
Rat Heart ◽  
Calcium Concentration ◽  
Microsomal Fraction ◽  
The Other ◽  
Heart Cell ◽  
Differential Centrifugation ◽  
Serum Calcium Concentration ◽  
Significant Prolongation ◽  
Normal Rat ◽  
Cell Fractions

Electrocardiographic observations were correlated with the 2- and 24-hr uptake of Ca45 by the myocardium of the normal rat and the rat injected subcutaneously with manganese chloride. The distribution of Ca45 in the heart cell was determined by differential centrifugation and the effects of manganese noted. Normally, about 40% of the Ca45 in the rat heart was found in the microsomal fraction, the remainder being distributed equally in the other cell fractions at 2 hr. None was found in the supernatant material after centrifuging for 17 hr. After manganese injection the total uptake of Ca45 was doubled at 2 hr due to increases in the microsomal and 17-hr fractions. Control values were re-established by 24 hr. Electrocardiograms showed a significant prolongation of the Q-T interval at 2 and 24 hr after manganese injection. There was a significant decrease in serum calcium concentration 24 hr after the injection of manganese.

Anion-stimulated ATPase in locust rectal epithelium

1988 ◽  
Vol 66 (2) ◽  
pp. 431-438 ◽  
Author(s):  
R. A. Lechleitner ◽  
J. E. Phillips
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Microsomal Fraction ◽  
Leucine Aminopeptidase ◽  
Mitochondrial Fraction ◽  
Mitochondrial Enzymes ◽  
Differential Centrifugation ◽  
Succinate Cytochrome C Reductase ◽  
Nacl Cotransport

The rectum, the main reabsorptive site in the locust excretory system, actively transports Cl−. This Cl− absorption is electrogenie, not dependent on Na+or [Formula: see text] and insensitive to inhibitors of NaCl cotransport or [Formula: see text] exchange. To determine if active Cl− transport across rectal epithelia might be due to an anion-stimulated ATPase, a microsomal fraction was obtained by differential centrifugation. Microsomal ATPase activity was stimulated in the following sequence: sulphite > bicarbonate > chloride. Maximal ATPase activity was obtained at 25 mM [Formula: see text] or 25 mM Cl−. Thiocyanate (10 mM) inhibited 90% of the anion-stimulated ATPase activity. The microsomal fraction was enriched in the plasma membrane markers, leucine aminopeptidase, alkaline phosphatase, 5′-nucleotidase, and γ-glutamyItranspeptidase, and had little contamination of the mitochondrial enzymes, succinate cytochrome c reductase and cytochrome oxidase. Na,K-ATPase was enriched in the mitochondrial fraction. Microscopic examination confirmed that basolateral membranes were associated with mitochondria following differential centrifugation, while the microsomal fraction contained little mitochondrial contamination. These results indicate the presence of an anion-stimulated ATPase activity that could be responsible for active Cl− transport across locust recta.

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.

scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1958 ◽  
Vol 4 (2) ◽  
pp. 203-218 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Electron Microscopy ◽  
Guinea Pig ◽  
Pancreatic Tissue ◽  
Supernatant Fraction ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Cytochemical Study ◽  
Specific Activities ◽  
Main Components ◽  
Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.

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

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.

Dynamic Transformation of Austenite at Temperatures above the Ae3

2018 ◽  
Vol 941 ◽  
pp. 633-638
Author(s):  
John Joseph Jonas ◽  
Clodualdo Aranas Jr. ◽  
Samuel F. Rodrigues
Keyword(s):  
Electron Microscopy ◽  
Driving Force ◽  
Energy Difference ◽  
Accelerated Cooling ◽  
Free Energy Difference ◽  
Dynamic Transformation ◽  
Plate Rolling ◽  
Temperature Transformation ◽  
Mn Steel ◽  
Kinetics Of

Under loading above the Ae3 temperature, austenite transforms displacively into Widmanstätten ferrite. Here the driving force for transformation is the net softening during the phase change while the obstacle consists of the free energy difference between austenite and ferrite as well as the work of shear accommodation and dilatation during the transformation. Once the driving force is higher than the obstacle, phase transformation occurs. This phenomenon was explored here by means of the optical and electron microscopy of a C-Mn steel deformed above their transformation temperatures. Strain-temperature-transformation (STT) curves are presented that accurately quantify the amount of dynamically formed ferrite; the kinetics of retransformation are also specified in the form of appropriate TTRT diagrams. This technique can be used to improve the models for transformation on accelerated cooling in strip and plate rolling.

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