NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Properties of the aromatase system associated with the mitochondrial fraction of human placenta

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35–50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16α-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

An intermediate of ubiquinone biosynthesis exists in the microsomal fraction of HepG2 cells

We analyzed lipids extracted from human hepatoma HepG2 cells using a high performance liquid chromatograph equipped with a reversed phase column and found a compound with a mass spectrum showing certain diagnostic ion fragments of 1-methoxy-5-polyprenyl-phenol, a known intermediate of ubiquinone biosynthesis. Universally radiolabeled [14C]-p-hydroxybenzoate, a precursor of ubiquinone, was incorporated into the compound on incubation with the cells, suggesting that the compound is a precursor of ubiquinone. The presence of the compound in the microsomal fraction of HepG2 cells was not due to contamination by the mitochondrial fraction because the activity of succinate-cytochrome c reductase in the microsomal fraction was below 1% of that in the mitochondrial fraction, whereas the contents of ubiquinone and the compound in the former were 4.6 and 7.8% of those in the latter, respectively. These results support the hypothesis that ubiquinone biosynthesis might occur in microsomes as well as mitochondria.     

Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes

Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction then declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Preparation of liver microsomes with high recovery of endoplasmic reticulum and a low grade of contamination

A modified procedure for preparing the microsomal fraction from rat liver was developed with the aim of increasing the recovery without increasing the degree of contamination. 87% of the membranes of the microsomal fraction isolated from the first mitochondrial (10 000 X g) supernatant originates from the endoplasmic reticulum, representing a 35% yield. By gentle resuspension of the 10 000 X g pellet followed by differential centrifugation a second crop of microsomes can be prepared which, together with the first crop, gives a 55% total recovery of microsomal markers. 87% of the protein in this second crop also originates from the endoplasmic reticulum and this fraction has properties similar to those of the first crop. Contaminating membranes include Golgi membranes (0.6% of the total protein), mitochondria (2.5%), lysosomes (5%) and plasma membranes (5%). Collecting further crops increases the contamination. Subfractionation studies revealed almost identical distributions of ribosome-rich, ribosome-poor and smooth membranes in the two crops of microsomal fractions. The results obtained after treatment of the animals with phenobarbital or methylcholantrene were similar to those obtained with control animals; but in the case of methylcholantrene treatment the second crop represents a larger portion of the total membranes of the endoplasmic reticulum.     

Characterization of dog cardiac microsomes Use of zonal centrifugation to fractionate fragmented sarcoplasmic reticulum, (Na + K)-activated ATPase and mitochondrial fragments

Cardiac microsomes represent a heterogeneous fraction which contains mitochondrial, plasma membrane and lysosomal enzymes in addition to markers believed to originate in the sarcoplasmic reticulum. The exact composition of this fraction depends on the method of preparation in that prolonged homogenization of ventricular myocardium increases both the yield of microsomal protein and the proportion of the mitochondrial contaminant.

Ultracentrifugation of cardiac microsomes on density gradients made with sucrose alone is of limited value in isolating fragmented sarcoplasmic reticulum. Because of aggregation of the microsomes, zonal ultracentrifugation in sucrose permits isolation of material with only slight enhancement in the activity of markers for the sarcoplasmic reticulum. In the presence of LiBr, used under conditions which inhibit the damaging effects of this salt on the activities studied, aggregation of the microsomal fraction is reduced and density gradient fractionation is more effective.

The fragmented sarcoplasmic reticulum prepared by zonal centrifugation in 0.5 M LiBr contains less than 1/5 the level of mitochondrial enzymes found in the original microsomes while the rate of Ca²⁺ uptake is enhanced 2-fold and the extent of Ca²⁺ uptake is enhanced 4-fold over that in the crude microsomal fraction. The sarcoplasmic reticulum markers were concentrated in a region of the gradient containing approx. 5% of the original protein that did not correspond to an obvious protein peak.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Pisum sativum seedlings

3-Hydroxy-3-methylglutaryl coenzyme A reductase from seedlings of Pisum sativum L. is localized in the plastids, mitochondria, and microsomes. Separation of the microsomal fraction into heavy and light subfractions shows that 95% of the microsomal activity is associated with the light subfraction. Definitive localization was achieved by showing that reductase activity comigrated with organelle markers on sucrose density gradients. Differential centrifugation studies showed that the microsomal fraction contained 80% of the total cellular activity, and the mitochondrial and plastid fractions each contained about 10%.The results suggest the existence of three parallel biosynthetic pathways which may be important in regulating the synthesis of isoprenoids characteristic of the individual organelles.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Subcellular distribution of aromatase in human placenta and ovary.

The aromatization of androstenedione in human ovarian microsomes is inhibited by an antibody to porcine hepatic microsomal NADPH-cytochrome c reductase. Likewise, the antibody inhibits aromatization in mitochondria isolated from human ovaries and placentae. A given quantity of the antibody produces the same percent inhibition of aromatization in microsomes and mitochondria of both ovaries and placentae. These data, in addition to the low specific activity observed for the mitochondrial aromatase, indicate that aromatization in mitochondria probably results from microsomal contamination.     

Subcellular distribution of thyroxine 5'-monodeiodinase activity in rabbit kidney

Thyroxine 5'-monodeiodinase is located in the proximal tubules of the rabbit kidney. To estimate the subcellular distribution of 5'-monodeiodinase activity, we prepared subcellular fractions, a basolateral membrane fraction and a brush border membrane fraction, from kidneys of Japanese white rabbits. Each fraction (0.5 mg protein) was incubated at 37 degrees C for 60 min with 0.5 micrograms T4 in the presence of 5 mM DTT. The T3 generated in the reaction mixture was extracted with cold ethanol and measured by RIA. For analysis of propylthiouracil-insensitive thyroxine 5'-monodeiodinase, we examined its kinetic behavior at nanomolar concentrations of the substrate, T4, in the presence of 100 microM propylthiouracil. In order of decreasing activity, basolateral membrane, microsomal fraction, mitochondrial fraction, cytosolic fraction, brush border membrane and nuclear fraction were capable of converting T4 to T3. Upon addition of 10(-5) M propylthiouracil to the reaction mixture, 5'-monodeiodinase activities of basolateral membrane and brush border membrane were inhibited by more than 90%, but that of microsomes was inhibited by only about 50%. In addition, kinetic analysis of microsomal 5'-monodeiodinase activity at nanomolar T4 concentrations in the presence of 10(-4) M propylthiouracil suggested on apparent Km of 3.8 nmol. These results indicate that there is high-Km 5'-monodeiodinase activity (PTU-sensitive) in the basolateral and brush border membranes and also high-Km and low-Km 5'-monodeiodinase (PTU-insensitive) in the microsomes of rabbit kidney.     

Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes

The aim of the present study was to characterize the acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity in human liver microsomes. Liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In 34 patients the enzyme activity of the microsomal fraction averaged 6.6 +/- 0.7 (mean +/- SEM) pmol.min-1.mg protein-1 in the absence of exogenous cholesterol. Freezing of the liver biopsy in liquid nitrogen increased the enzyme activity five- to sixfold. Similarly, freezing of the microsomal fraction prepared from unfrozen liver tissue increased the enzyme activity about twofold. These results may help to explain previous disparate results reported in the literature. The enhanced ACAT activity obtained by freezing was at least partly explained by a transfer of unesterified cholesterol to the microsomal fraction and possibly also by making the substrate(s) more available to the enzyme. Preincubation of the microsomal fraction, prepared from unfrozen liver tissue, with unlabeled cholesterol increased the enzyme activity about fivefold. This finding indicates that hepatic ACAT in humans can also utilize exogenous cholesterol as substrate. Addition of cholesterol to frozen microsomes prepared from unfrozen liver tissue increased the ACAT activity two- to threefold, whereas addition of cholesterol to microsomes prepared from frozen liver tissue did not further increase the enzyme activity. No evidence supporting the concept that ACAT is activated-inactivated by phosphorylation-dephosphorylation could be obtained by assaying the enzyme under conditions similar to those during which the human HMG-CoA reductase is inactivated-activated.     

Rat lung carbonic anhydrase: activity, localization, and isozymes

Carbonic anhydrase activity in rat lungs perfused free of blood was localized by homogenization of the tissue followed by differential centrifugation. Four fractions were obtained from the homogenate, a cell debris pellet with a mitochondrial pellet and a microsomal pellet with a clear cytosol supernatant. The last named fraction contained 67% of the total enzyme activity; the cell debris contained 18%, and the mitochondrial and microsomal contained 8 and 7%, respectively. Of the 33% of enzyme activity associated with the pellet fraction, 25% could be experimentally defined as membrane associated by its solubilization with 0.3 M tris-(hydroxymethyl) aminoethane sulfate buffer. The remainder was defined as membrane bound. Purification of the soluble carbonic anhydrase from the lung yielded two isozymes with electrophoretic and inhibitor sensitivities apparently identical with the blood isozymes. Hemoglobin analysis showed that the lung isozymes could not have included more than 0.03% enzyme from blood contamination. The carbonic anhydrase activity present in the whole rat lung would give an average acceleration of the CO2 hydration reaction under physiological conditions over the uncatalyzed rate of 122, sufficient to maintain equilibration between CO2 and plasma HCO3- during blood transit of the lung. If the membrane-associated activity is mostly on the plasma membrane of the endothelial cells and available to the capillary blood, it would be sufficient to give this acceleration. We suggest that the possible source of this membrane-associated activity might be adsorption from the blood of carbonic anhydrase liberated by erythrocyte lysis.     

Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: the olfactory bulb and the mitochondrial fraction have high levels of activity

In brain of female monkey (M. fascicularis) the content of cytochrome P-450 and benzo(a)pyrene hydroxylase activity in the mitochondrial fraction exceeded that of the microsomes by more than 4-fold. The mitochondrial drug metabolism activity exhibited substrate specificity and, unlike the microsomes, did not catalyze 7-ethoxyresorufin O-deethylase reaction. Moreover, the rate of benzo(a)pyrene hydroxylase activity by both the mitochondrial and the microsomal fractions displayed regional variation with the olfactory bulb displaying the highest hydroxylase activity. In contrast, the microsomal 7-ethoxyresorufin O-deethylase activity was uniformally distributed in all brain regions.     

ACTIVE UPTAKE OF 45Ca BY A MICROSOMAL FRACTION PREPARED FROM RAT DORSAL ROOTS

Abstract— A microsomal fraction prepared from rat dorsal spinal nerve roots accumulated ⁴⁵Ca by a temperature–and ATP-dependent mechanism. Uptake, which was maximal at pH 7.2–7.4, was potentiated 4-fold by 8 m m -oxalate and was linear over a 20 min incubation period. Ca uptake was not inhibited by sodium azide or by oligomycin and only slightly by ruthenium red suggesting that it was not of mitochondrial origin. On a sucrose density gradient the microsomal fraction equilibrated at between 0.25 m - and 0.65 m -sucrose but, using a discontinuous gradient over this range, no fraction enriched in Ca-accumulating activity could be separated. The possibility is discussed that the Ca-accumulating microsomes may be derived from smooth endoplasmic reticulum.     

Ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta (L.) Midgut: Is the dual localization real?

The dual localization of ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta larval midgut was investigated. Cosubstrate requirements and response to osmolarity of the microsomal ecdysone 20-monooxygenase system were found to be different from those previously reported for the mitochondrial enzyme system. The microsomal monooxygenase utilized NADPH and, less efficiently, NADH as cosubstrates. NADPH and NADH effects were neither additive nor synergistic. NADPH yielded identical activities in isotonic and hypotonic incubations. Mitochondria and microsomes showed no synergistic interaction for ecdysone 20-hydroxylation. After washing of the mitochondria, a large proportion of their ecdysone 20-monooxygenase activity was lost. The extent of the loss was inversely correlated to the concentration of mitochondria in the incubation mixture. The addition of bovine serum albumin to the incubations (2 mg/ml) largely restored the original activities. The microsomal contamination in mitochondrial pellets after each of three successive washings was determined by measuring the activity of a microsomal marker enzyme, NADPH-cytochrome c reductase. At each step of the purification, the ecdysone 20-monooxgenase activity of the mitochondrial preparations far exceeded the activity attributable to the microsomal contamination. These results confirm the existence of two independent ecdysone 20-monooxygenase systems in the midgut of M. sexta larvae.     

Hydroxymethylglutaryl coenzyme A reductase activity of adult Hymenolepis diminuta

The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestode's cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestode's HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.