Enzyme and phospholipid asymmetry in liver microsomal membranes.

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.

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Influence of dietary fat on the lipid composition of rat brain synaptosomal and microsomal membranes

Biochemical Journal ◽

10.1042/bj2080631 ◽

1982 ◽

Vol 208 (3) ◽

pp. 631-640 ◽

Cited By ~ 101

Author(s):

M Foot ◽

T F Cruz ◽

M T Clandinin

Keyword(s):

Fatty Acids ◽

Rat Brain ◽

Lipid Composition ◽

Dietary Fat ◽

Monounsaturated Fatty Acids ◽

Microsomal Membrane ◽

Omega 3 ◽

Membrane Composition ◽

Omega 6 ◽

Microsomal Membranes

The modulation of rat brain microsomal and synaptosomal membrane lipid by diet fat was examined. Brain synaptosomal and microsomal membrane composition was compared for rats fed on diets containing either soya-bean oil (SBO), SBO plus choline, SBO lecithin, sunflower oil (SFO), chow or low-erucic acid rape-seed oil (LER) for 24 days. Cholesterol and phosphatidylcholine levels in both membranes were altered by diet. Diet fat also affected the microsomal content of sphingomyelin. Change in membrane phosphatidylcholine level was related to the relative balance of omega-6, omega-3 and monounsaturated fatty acids within the diets fed. The highest phosphatidylcholine levels appeared in membranes of animals fed on SBO lecithin and the lowest in those fed on LER. Microsomal membrane cholesterol and sphingomyelin content increased by feeding on SBO lecithin. In both synaptosomal and microsomal membranes a highly significant correlation was observed between membrane phosphatidylcholine and cholesterol content. The fatty acyl composition of phospholipids from both membranes also altered with diet and age. Alteration in fatty acid composition was observed in response to dietary levels of omega-6, omega-3 and monounsaturated fatty acids, but the unsaturation index of each phospholipid remained constant for all diet treatments. These changes in lipid composition suggest that dietary fat may be a significant modulator in vivo of the physicobiochemical properties of brain synaptosomal and microsomal membranes.

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Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

Biochemical Journal ◽

10.1042/bj1900737 ◽

1980 ◽

Vol 190 (3) ◽

pp. 737-746 ◽

Cited By ~ 15

Author(s):

Michael B. Cooper ◽

John A. Craft ◽

Margaret R. Estall ◽

Brian R. Rabin

Keyword(s):

Endoplasmic Reticulum ◽

Cytochrome C ◽

Rat Liver ◽

Smooth Endoplasmic Reticulum ◽

Reductase Activity ◽

Asymmetric Distribution ◽

Trypsin Treatment ◽

Cytochrome C Reductase ◽

Nadph Cytochrome ◽

Cytochrome P 450

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).

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Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.)

Biochemical Journal ◽

10.1042/bj2870141 ◽

1992 ◽

Vol 287 (1) ◽

pp. 141-144 ◽

Cited By ~ 57

Author(s):

M A Smith ◽

L Jonsson ◽

S Stymne ◽

K Stobart

Keyword(s):

Ricinoleic Acid ◽

Castor Bean ◽

Ricinus Communis ◽

Polyclonal Antibodies ◽

Reductase Activity ◽

Cytochrome B5 ◽

Hydroxylase Activity ◽

Ricinus Communis L ◽

Microsomal Membranes ◽

Cytochrome P 450

The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved.

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Vitamin K antagonism of coumarin anticoagulation. A dehydrogenase pathway in rat liver is responsible for the antagonistic effect

Biochemical Journal ◽

10.1042/bj2360685 ◽

1986 ◽

Vol 236 (3) ◽

pp. 685-693 ◽

Cited By ~ 35

Author(s):

R Wallin

Keyword(s):

Rat Liver ◽

Vitamin K ◽

Cytochrome B5 ◽

Test System ◽

Antagonistic Effect ◽

Anticoagulant Drugs ◽

Dt Diaphorase ◽

Cytochrome P 450 ◽

Important Enzyme

In the liver, it appears that there are two different pathways for vitamin K reduction. One pathway is irreversibly inhibited by coumarin anticoagulant drugs. The other pathway has been shown in the present study to be composed of enzymes that are not effected by physiological ‘in vivo’ concentrations of these drugs. This pathway appears to be responsible for the antidotal effect of vitamin K in overcoming coumarin poisoning. In rat liver the pathway has been shown to be composed of DT-diaphorase (EC.1.6.99.2) and a microsomal dehydrogenase(s). The activity of the microsomal dehydrogenase(s) was 3.6-fold higher with NADH than with NADPH present in the test system. It appears that this enzyme is the physiologically important enzyme in the pathway. In contrast with DT-diaphorase, this enzyme(s) is shown to be tightly associated with the mirosomal membrane. The enzyme(s) is not identical with either of the quinone-reducing enzymes cytochrome P-450 reductase or cytochrome-b5 reductase. Our data thus postulate the existence of an as-yet-unidentified microsomal dehydrogenase that appears to have an important function in the pathway.

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