SOLUBILIZATION OF THYROXINE-5′-DEIODINASE ACTIVITY FROM RAT LIVER MICROSOME FRACTION

1979 ◽  
Vol 92 (4) ◽  
pp. 694-701 ◽  
Author(s):  
Masaru Takaishi ◽  
Taeko Shimizu ◽  
Yoshimasa Shishiba
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
Rat Liver ◽  
Specific Activity ◽  
Cytochrome B5 ◽  
Microsomal Membrane ◽  
Cytochrome C Reductase ◽  
Deiodinase Activity ◽  
Microsome Fraction ◽  
Nadh Cytochrome

ABSTRACT The greater part of T3 is converted from T4 in liver or kidney. The majority of this activity exists in microsomal fraction. In the present study, we investigated whether this activity can be solubilized from rat liver microsomal pellet with various concentrations of deoxycholate (DOC). The extent of solubilization was compared with that of protein, rotenone insensitive NADH cytochrome c reductase or NADH cytochrome b5 reductase, which have been shown to associate with microsomal membrane rather than luminar contents. When 0.05 % of DOC which was capable of releasing luminar contents of microsomal vesicles was applied to microsomal suspension, only a limited part of NADH cytochrome b5 reductase, rotenone insensitive NADH cytochrome c reductase or T4-5′-deiodinase activity was solubilized. When the concentration of DOC was increased to 0.125 %, 41 % of T4-5′-deiodinase activity was solubilized. Solubilization of protein, NADH cytochrome b5 reductase or rotenone insensitive NADH cytochrome c reductase was increased abruptly to 66 %, 58 % or 63%, respectively. The highest specific activity was obtained at 0.125% DOC. These results suggest that the T4-5′-deiodinase is associated with microsomal membrane instead of luminar contents.

scholarly journals Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex.

1980 ◽  
Vol 85 (3) ◽  
pp. 501-515 ◽  
Author(s):  
N Borgese ◽  
J Meldolesi
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Golgi Complex ◽  
Cytochrome B5 ◽  
Liver Cells ◽  
Cytochrome C Reductase ◽  
Cytochrome B5 Reductase ◽  
Galactosyl Transferase ◽  
Rat Liver Cells ◽  
Nadh Cytochrome

The subcellular distribution of NADH-cytochrome b5 reductase in rat liver cells was reinvestigated. In fresh heavy and light Golgi fractions (GF3 and GF1 + 2) and in mitochondria, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was approximately 100, 60, and 30%, respectively, of the value found in microsomes. However, the Golgi enzyme was unstable inasmuch as pelleting and resuspending the fresh fractions resulted in a considerable inactivation (40--60%), which was further increased with subsequent storage at 4 degrees C. A similar inactivation was observed using cytochrome b5 but not ferricyanide as electron acceptor. The inactivation of Golgi NADH-cytochrome c reductase activity was independent of the protein concentration of the fractions during storage, was unaffected by the addition of the antioxidant butylated hydroxytoluene, but was partly prevented by buffering the fractions at neutral pH and by storage at--20 degrees C. A total Golgi fraction was analyzed by density equilibration on continuous sucrose gradients after exposure to digitonin. As expected, the distribution of both protein and galactosyl transferase were shifted to higher densities by this treatment. However, not all galactosyl transferase-bearing elements were shifted to the same extent by exposure to the detergent, suggesting a biochemical heterogeneity of the Golgi complex. In contrast to their behavior in microsomes, the distribution of NADH-cytochrome c reductase and cytochrome b5 of Golgi fractions was shifted by digitonin, although to a lesser extent than that of galactosyl transferase. These results indicate that NADH-cytochrome b5 reductase is an authentic component of Golgi membranes, as well as of microsomes and of mitochondria. The conflicting results reported in the past on the Golgi localization of the enzyme could be due, on the one hand, to the differential lability of the activity in its various subcellular locations and, on the other, to the heterogeneity of the Golgi complex in terms of both cholesterol and enzyme distribution.

scholarly journals Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride

1979 ◽  
Vol 178 (2) ◽  
pp. 323-329 ◽  
Author(s):  
Y Hino ◽  
S Minakami
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Cytochrome B5 ◽  
Transport Pathway ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Haem Oxygenase ◽  
Reported Data ◽  
Nadph Cytochrome C Reductase

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH–cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.

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

1974 ◽  
Vol 61 (1) ◽  
pp. 213-231 ◽  
Author(s):  
Henri Beaufay ◽  
Alain Amar-Costesec ◽  
Denise Thinès-Sempoux ◽  
Maurice Wibo ◽  
Mariette Robbi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Cytochrome C ◽  
Monoamine Oxidase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Cytochrome B5 ◽  
Microsomal Protein ◽  
Cytochrome C Reductase ◽  
Group A ◽  
Group B

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, ß-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.

scholarly journals AN ELECTRON-TRANSPORT SYSTEM ASSOCIATED WITH THE OUTER MEMBRANE OF LIVER MITOCHONDRIA

1967 ◽  
Vol 32 (2) ◽  
pp. 415-438 ◽  
Author(s):  
Gian Luigi Sottocasa ◽  
Bo Kuylenstierna ◽  
Lars Ernster ◽  
Anders Bergstrand
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Outer Membrane ◽  
Respiratory Chain ◽  
Transport System ◽  
Cytochrome B5 ◽  
Liver Mitochondria ◽  
Electron Transport System ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b5 reductase and cytochrome b5.

scholarly journals Membrane-bound redox proteins of the murine Friend virus-induced erythroleukemia cell.

1979 ◽  
Vol 83 (1) ◽  
pp. 231-239 ◽  
Author(s):  
S R Slaughter ◽  
D E Hultquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Dimethyl Sulfoxide ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Erythroid Cells ◽  
Friend Virus ◽  
Cytochrome C Reductase ◽  
Membrane Bound ◽  
Nadh Cytochrome

We have obtained and studied a 105,000-g pellet from T-3-Cl-2 cells, a cloned line of Friend virus-induced erythroleukemia cells. By difference spectrophotometry, the pellet was shown to contain cytochrome b5 and cytochrome P-450, hemeproteins that have been shown to participate in electron-transport reactions of endoplasmic reticulum and other membranous fractions of various tissues. The pellet also possesses NADH-cytochrome c reductase activity which is inhibited by anti-cytochrome b5 gamma-globulin, indicating the presence of cytochrome b5 reductase. This is the first demonstration of membrane-bound forms of these redox proteins in erythroid cells. Dimethyl sulfoxide-treated T-3-Cl-2 cells were also shown to possess membrane-bound cytochrome b5 and NADH-cytochrome c reductase activity. We failed to detect soluble cytochrome b5 in the 105,000-g supernatant fraction from homogenates of untreated or dimethyl sulfoxide-treated T-3-Cl-2 cells. In contrast, erythrocytes obtained from mouse blood were shown to possess soluble cytochrome b5 but no membrane-bound form of this protein. These findings are supportive of our hypothesis that soluble cytochrome b5 of erythrocytes is derived from endoplasmic reticulum or some other membrane structure of immature erythroid cells during cell maturation.

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

1983 ◽  
Vol 244 (5) ◽  
pp. G480-G490 ◽  
Author(s):  
A. Kribben ◽  
T. Tyrakowski ◽  
I. Schulz
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Tissue Homogenate ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.

Thyroxine stimulation of rat liver microsomal NADH-cytochrome c reductase in vitro

Life Sciences ◽  
1974 ◽  
Vol 15 (12) ◽  
pp. 2059-2068 ◽  
Author(s):  
Fred H. Faas ◽  
William J. Carter ◽  
James O. Wynn
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome ◽  
Stimulation Of
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