Apparent saturability of a 4S dihydrotestosterone-binding protein in rat muscle cytosol: role of 3α-hydroxysteroid dehydrogenase and albumin

1977 ◽  
Vol 55 (9) ◽  
pp. 995-1000 ◽  
Author(s):  
France T. Dionne ◽  
Jean Y. Dube ◽  
Roland R. Tremblay
Keyword(s):  
Enzymatic Activity ◽  
Ammonium Sulfate ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Rat Serum ◽  
Binding Behaviour

In our studies of the binding of steroids in rat skeletal muscles, we have shown the existence of a saturable '4S' dihydrotestosterone-binding protein of low affinity (Kd = 1.16 × 10−6 M). Competition studies led us to believe that the binding of dihydrotesterone (DHT) was due to 3α-hydroxysteroid dehydrogenase (3α-OHSD) enzyme responsible for the conversion of DHT to androstanediol (Adiol). Indeed, both the binding and the enzymatic activity are inhibited by various 3-keto steroids. In addition, the dissociation constant (Kd) and the Michaelis constant (Km) of the enzyme are similar. However, experiments with ammonium sulfate fractions of the cytosol have shown a partial separation of the binding and of the enzymatic activity. On the other hand, we have established that DHT (3 μM) is almost completely metabolized to Adiol during a 2-h incubation at 0 °C even in the absence of added coenzymes. Furthermore the '4S' protein binds Adiol more strongly than DHT and this binding is not saturable. Finally the binding behaviour of both DHT and Adiol with either muscle cytosol or rat albumin is similar when subjected to ammonium sulfate fractionation and sucrose density gradient centrifugation. In conclusion, the skeletal muscle 3α-OHSD rapidly metabolizes DHT into Adiol which then binds strongly to a nonspecific binding protein, presumably rat serum albumin. Thus it can be said that the observed saturability of DHT binding is only apparent.

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
Author(s):  
A. R. EASTMAN ◽  
A. M. NEVILLE
Keyword(s):  
Molecular Weight ◽  
Adrenal Glands ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Molecular Sizes ◽  
Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

SUBCELLULAR LOCALIZATION OF NEUROTRANSMITTERS AND RELEASING FACTORS IN THE RAT MEDIAN EMINENCE

1970 ◽  
Vol 48 (2) ◽  
pp. 205-213 ◽  
Author(s):  
F. CLEMENTI ◽  
B. CECCARELLI ◽  
E. CERATI ◽  
M. L. DEMONTE ◽  
M. FELICI ◽  
...  
Keyword(s):  
Median Eminence ◽  
Gradient Centrifugation ◽  
Physiological Role ◽  
Sucrose Density Gradient ◽  
Nerve Terminals ◽  
Sucrose Density Gradient Centrifugation ◽  
Different Types ◽  
Releasing Factors ◽  
Electron Microscopical

SUMMARY The neurohaemal part of the median eminence of the rat hypothalamus is characterized by numerous nerve terminals which end near a rich network of fenestrated capillaries. An attempt was made to isolate different types of nerve terminals by means of sucrose density-gradient centrifugation. The subcellular fractions obtained were assayed for dopamine, noradrenaline and 5-hydroxytryptamine. In addition FSH- and GH-releasing activities were determined. A sample of each fraction obtained was taken for electron microscopical observations. Dopamine, noradrenaline, 5-hydroxytryptamine, GH- and FSH-releasing factors were present in higher concentration in the nerve endings. A further fractionation showed that noradrenaline was present in the lightest synaptosomal band, dopamine in the middle one, and 5-hydroxytryptamine in the heaviest. GH-RF and FSH-RF were recovered mainly from the band containing dopamine. The relevance of this localization to the physiological role of the median eminence is discussed.

scholarly journals Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress

2020 ◽  
Vol 21 (17) ◽  
pp. 6146
Author(s):  
Yuka Eura ◽  
Toshiyuki Miyata ◽  
Koichi Kokame
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Dependent Manner ◽  
Dynamic Nature ◽  
Sucrose Density Gradient Centrifugation

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

scholarly journals Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells.

1984 ◽  
Vol 98 (1) ◽  
pp. 179-187 ◽  
Author(s):  
G Jung ◽  
R M Helm ◽  
C A Carraway ◽  
K L Carraway
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mammary Adenocarcinoma ◽  
Con A ◽  
Sucrose Density Gradient Centrifugation ◽  
Adenocarcinoma Cells ◽  
Triton X

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.

Studies on 2′,3′-cyclic nucleotide-3′-phosphohydrolase from brain

1969 ◽  
Vol 47 (10) ◽  
pp. 961-966 ◽  
Author(s):  
Robert W. Olafson ◽  
George I. Drummond ◽  
Jack F. Lee
Keyword(s):  
Density Gradient ◽  
Cyclic Nucleotide ◽  
Gradient Centrifugation ◽  
Physiological Role ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain White Matter ◽  
Cyclic Phosphate

Studies on a 2′,3′-cyclic nucleotide-3′-phosphohydrolase are presented. Bovine brain white matter was fractionated by differential centrifugation and sucrose density gradient techniques. The phosphohydrolase was distributed throughout all primary fractions: nuclear, mitochondrial, and microsomal. When these fractions were subjected to sucrose density gradient centrifugation the enzyme appeared only in the myelin-containing layers. Brains of newborn rats were essentially devoid of phosphohydrolase activity and the content rose rapidly during the 12th and 25th days, coinciding precisely with the development of myelin. Mutant 'quaking' mice whose brains are partially deficient in myelin were also partially deficient in enzyme activity. The data provide evidence that the phosphohydrolase is associated with myelin.The enzyme opens the cyclic phosphodiester linkage of Ap(Ap)2A cyclic P and Ap(Ap)6A cyclic P without rupture of internucleotide bonds. The enzyme also hydrolyzed the 2′,3′-cyclic phosphorothioates of uridine and guanosine. The Km for adenosine 2′,3′-cyclic phosphate was determined to be 1.9 × 10−3 M, an inordinately high value, and since nothing is known about the physiological role of the enzyme, the possibility is raised that the true substrate may not be a cyclic nucleotide.

Cl--HCO3--ATPase in gills of the rainbow trout: evidence for its microsomal localization

1980 ◽  
Vol 238 (3) ◽  
pp. R251-R259
Author(s):  
M. Bornancin ◽  
G. de Renzis ◽  
R. Naon
Keyword(s):  
Rainbow Trout ◽  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Salmo Gairdneri ◽  
Acid Base Balance ◽  
Sucrose Density Gradient Centrifugation ◽  
Base Balance

The intracellular localization of a Cl--HCO3--ATPase, which is inhibited by SCN-, was studied in the gills of the rainbow trout, Salmo gairdneri. This activity can be measured in the absence of contamination by mitochondria (i.e., in the absence of succinate dehydrogenase or cytochrome c oxidase activities). The distribution of the 5'-nucleotidase and of the ATPase stimulated by Cl- and HCO3- after sucrose density gradient centrifugation of the microsomal fraction was compared. Because those activities cannot be separated, it is postulated that the anion-stimulated ATPase is located in the plasma membrane. The activation of this microsomal anion ATPase by chloride has been studied extensively. The possible role of the Cl--HCO3--ATPase of trout gills in the Cl-/HCO3- exchange and in the regulation of the internal acid-base balance is discussed.

Ox Brain Aminopeptidases, Further Purification of Three Bovine Enzymes

1971 ◽  
Vol 49 (6) ◽  
pp. 676-685 ◽  
Author(s):  
R. E. Sobel ◽  
A. S. Brecher
Keyword(s):  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Heat Stability ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Supernatant Fraction ◽  
Differential Heat ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain Extracts

Four peptidases capable of acting upon Leu∙Gly∙Gly have been separated from bovine brain extracts, and three have been highly purified by ammonium sulfate fractionation and by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. A Mn2+-stimulated aminopeptidase (E2) was purified 180-fold over an initial 16 300 × g supernatant fraction and has a M.W. of approximately 117 250 as estimated by sucrose density gradient centrifugation. Two peptidases which are inhibited by heavy metals (E1P1 and E1P2) were purified approximately 2683-fold and 2461-fold over the initial extracts and have approximate M.W.'s of 61 500 and 85 700, respectively. E1P1 and E1P2 have Km's of 0.36 and 0.38 mM, respectively. The pH optima for E1P1, E1P2, and E2 are 6.6–7.8, 7.2–8.1, and 7.8–9.6. E2 is more heat-labile than E1P1 and E1P2 since it is completely inactivated by heating at 55° for 30 min at pH 6.5 under which conditions the latter two enzymes retain full activity. The ΔEa for the hydrolysis of Leu∙Gly∙Gly was calculated to be 10 800, 13 420, and 15 480 for E1P1, E1P2, and E2. E1P1 and E1P2 hydrolyze tripeptides but do not cleave dipeptides. E2 cleaves tripeptides, and only cleaves Leu∙Gly, of a series of dipeptides examined, having a specificity differing notably from leucine aminopeptidase. All the enzymes were inactive towards leucine-p-nitroanilide and leucine-β-naphthylamide, suggesting that they should not be classed among the arylamidases. On the basis of differential heat stability, it is estimated that a peptidase sedimented by ammonium sulfate at 20% saturation contains 40% of the original tripeptidase activity; E2 represents 30% of the activity; E1P1 and E1P2 contribute 12 and 18%, respectively, to the tripeptidase activity in the original extracts.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

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