Biosynthesis of Phosphatidic Acid in Mitochondria and Microsomes Via the Acylation of sn-Glycero-3-phosphate

1972 ◽  
Vol 50 (8) ◽  
pp. 936-948 ◽  
Author(s):  
J. B. Davidson ◽  
N. Z. Stanacev
Keyword(s):  
Phosphatidic Acid ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Marker Enzymes ◽  
Acyltransferase Activity ◽  
Isolated Mitochondria ◽  
Direct Acylation ◽  
Submitochondrial Fractions ◽  
Nadph Cytochrome C Reductase

Nuclei-free homogenate, prepared from guinea pig livers, was fractionated into subcellular particles which were then examined for the activities of two microsomal marker enzymes, glucose-6-phosphatase and NADPH: cytochrome c reductase. In an incubation system containing sn-glycero-3-phosphate, fatty acid, and various cofactors the intracellular distribution of acyl-CoA: sn-glycero-3-phosphate acyltransferase(s) was studied and compared with the distribution of the two microsomal marker enzymes.Results obtained showed that the highest specific activity for the acylation of sn-glycero-3-phosphate was associated with the microsomal fraction and the activity in each subcellular fraction paralleled activities of the two microsomal marker enzymes. Furthermore, the amount of acyl-CoA: sn-glycero-3-phosphate acyltransferase activity observed in the mitochondrial and submitochondrial fractions could be accounted for by the content of endoplasmic reticulum as determined by the marker enzymes. This observation was also true for brain, heart, and kidney, as well as for rat liver.These results are interpreted as evidence that isolated mitochondria are unable to synthesize phosphatidic acid by direct acylation of sn-glycero-3-phosphate.

Evidence for the Biosynthetic Difference between Isolated Mitochondria and Microsomes from Guinea-Pig and Rat Liver Regarding Lysophospharidic Acid, Phosphatidic Acid, CDP-Diglyceride, Phosphatidylglycerol, and Cardiolipin

1974 ◽  
Vol 52 (10) ◽  
pp. 936-939 ◽  
Author(s):  
J. B. Davidson ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Marker Enzymes ◽  
Synthetic Enzymes ◽  
Isolated Mitochondria ◽  
Significant Difference ◽  
Nadph Cytochrome C Reductase

The enzymatic activities of marker enzymes (NADPH – cytochrome c reductase and glucose-6-phosphatase) and synthetic enzymes (acyl-CoA:sn-glycero-3-phosphate acyltransferase, CTP:sn-3-phosphatidic acid cytidyltransferase, and CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase) were measured in both isolated mitochondria and microsomes from liver of guinea pig and rat. Results thus obtained show a significant difference in activities of these enzymes between subcellular particles within species and between two examined species. The activity of acyl-CoA:glycero-3-phosphate acyltransferase in guinea-pig mitochondria parallels the activity of microsomal marker enzymes in this fraction, while in rat liver mitochondria the activity is relatively higher and cannot be accounted for by the microsomal content as determined by marker enzymes. Implications of these results regarding mitochondrial autonomy in the biosynthesis of polyglycero-phosphatides and their precursors are discussed.

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

1974 ◽  
Vol 61 (1) ◽  
pp. 201-212 ◽  
Author(s):  
Alain Amar-Costesec ◽  
Henri Beaufay ◽  
Maurice Wibo ◽  
Denise Thinès-Sempoux ◽  
Ernest Feytmans ◽  
...  
Keyword(s):  
Analytical Study ◽  
Microsomal Fraction ◽  
Chemical Constituents ◽  
Microsomal Protein ◽  
High Yield ◽  
Marker Enzymes ◽  
Cytoplasmic Granules ◽  
Liver Homogenates ◽  
Nadph Cytochrome C Reductase ◽  
Phosphatase Alkaline

Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-ß-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.

scholarly journals Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1965 ◽  
Vol 97 (2) ◽  
pp. 389-392 ◽  
Author(s):  
G Vaes ◽  
P Jacques
Keyword(s):  
Cytochrome Oxidase ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Distribution Patterns ◽  
Mitochondrial Fraction ◽  
Acid Hydrolases ◽  
Mitochondrial Fractions ◽  
Tissue Homogenates ◽  
Isopycnic Centrifugation

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.

Localization of Phosphate-Independent Glutaminase in the Brush Border of Rat Kidney Cortex

1974 ◽  
Vol 52 (9) ◽  
pp. 762-766 ◽  
Author(s):  
J. Kalra ◽  
John T. Brosnan
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Rat Kidney ◽  
Sucrose Density Gradient ◽  
Kidney Cortex ◽  
Salt Treatment ◽  
Rat Kidney Cortex ◽  
Nadph Cytochrome C Reductase

A microsomal fraction that contains the highly enriched activities of NADPH – cytochrome c reductase, 5′-nucleotidase, phosphate-independent glutaminase, and alkaline phosphatase was isolated by differential centrifugation from rat kidney cortex. Continuous sucrose density gradient studies on this fraction have shown that the distribution pattern of phosphate-independent glutaminase is identical with that of alkaline phosphatase and the specific activity of these enzymes in peak fractions were 13- to 17-fold higher than in the whole homogenate. These results indicate that the phosphate-independent glutaminase is localized in the brush border of rat kidney cortex. The enzyme is truly membranous as it could not be removed by sonication, salt treatment, or pH alterations.

scholarly journals Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

1974 ◽  
Vol 144 (2) ◽  
pp. 265-275 ◽  
Author(s):  
G S Cobon ◽  
P D Crowfoot ◽  
A W Linnane
Keyword(s):  
Phosphatidic Acid ◽  
Microsomal Fraction ◽  
De Novo ◽  
Specific Activity ◽  
Neutral Lipids ◽  
Mitochondrial Fraction ◽  
Biogenesis Of Mitochondria ◽  
Glycerolipid Synthesis ◽  
Phosphatidylcholine Synthesis

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-3H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-14C]methionine and CDP-[Me-14C]choline appeared to be localized in the microsomal fraction.

scholarly journals Intramitochondrial localization of the 4-aminobutyrate-2-oxoglutarate transaminase from ox brain

1977 ◽  
Vol 162 (2) ◽  
pp. 303-307 ◽  
Author(s):  
I Schousboe ◽  
B Bro ◽  
A Schousboe
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Large Amplitude ◽  
Mitochondrial Membrane ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Outer Membranes ◽  
Submitochondrial Fractions

In order to determne the intramitochondrial location of 4-aminobutyrate transaminase, mitochondria were prepared from ox brain and freed from myelin and synaptosomes by using conventional density-gradient-centrifugation techniques, and the purity was checked electron-microscopically. Inner and outer membranes and matrix were prepared from the mitochondria by large-amplitude swelling and subsequent density-grient centrifugation. The fractions were characterized by using both electron microscopy and different marker enzymes. From the specific activity of the 4-aminobutyrate transaminase in the submitochondrial fractions it was concluded that this enzyme is associated with the innter mitochondrial membrane.

Cellular distribution of enzymes involved in phosphatidylcholine synthesis in developing rat lung

1983 ◽  
Vol 61 (2-3) ◽  
pp. 107-114 ◽  
Author(s):  
Frank Chan ◽  
Paul G. R. Harding ◽  
Tanya Wong ◽  
G. Frazer Fellows ◽  
Fred Possmayer
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Perinatal Period ◽  
Rat Lung ◽  
Developmental Profile ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Choline Incorporation ◽  
Developing Rat ◽  
Phosphatidylcholine Synthesis ◽  
Nadph Cytochrome C Reductase

The incorporation of radioactive choline into phosphatidylcholine and disaturated phosphatidylcholine in rat lung slices increased markedly before term and peaked after birth. The specific activity of cholinephosphate cytidylyltransferase in the microsomal fraction increased before birth but fell after delivery. The specific activity of this enzyme in the cytosol showed a marked increase at birth. The developmental profile for the total cytosolic activity per gram lung was similar to the pattern observed with choline incorporation. Although the specific activity of cholinephosphotransferase in the whole homogenate remained relatively constant throughout pulmonary maturation, there was a marked increase in the specific activity of this enzyme in the microsomal fraction at term. Similar findings were obtained with the microsomal marker NADPH-cytochrome c reductase. The basis of this disparity in specific activity profiles is being investigated further. The specific activity of lysophosphatidylcholine: lysophosphatidylcholine transacylase in rat lung homogenates increased during gestation but rose a further 10-fold between day 3 after birth and the adult. The specific activity of lysophosphatidylcholine: palmitoyl-CoA acyltransferase remained relatively constant throughout development. At term, the specific activity of the acylation enzyme was 10- to 15-fold greater than the specific activity of the transacylation enzyme. These observations are consistent with previous studies indicating that the accumulation of phosphatidylcholine and dipalmitoyl phosphatidylcholine during the perinatal period may be due to alterations in the activity of cholinephosphate cytidylyltransferase. Cholinephosphotransferase could also play a regulatory role. The formation of dipalmitoyl phosphatidylcholine appears to occur via the acylation, rather than the transacylation pathway.

In vitro 19-norandrogen synthesis by equine placenta requires the participation of aromatase

1995 ◽  
Vol 144 (3) ◽  
pp. 517-525 ◽  
Author(s):  
S Moslemi ◽  
P Silberzahn ◽  
J-L Gaillard
Keyword(s):  
Silica Gel ◽  
Aromatase Inhibitors ◽  
Column Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Term Placenta ◽  
Flow Detector

Abstract Explants of equine full-term placenta have been shown to synthesize 19-norandrogens from labelled androgens. Steroid metabolites were purified by silica-gel column chromatography then analysed and quantified by C18-reverse-phase HPLC coupled to a radioactive flow detector. 19-Norandrostenedione was subsequently recrystallized to constant specific activity, providing unequivocal evidence of its synthesis by the equine placenta. 19-Norandrostenedione synthesis appeared to be localized in the microsomal fraction. Regardless of the substrate used, formation of 19-norandrogens was far weaker than that of oestrogens; moreover, the yield of 17-oxosteroids produced was much greater than that of 17β-hydroxysteroids, suggesting the presence of a dehydrogenase with predominant oxidative activity. Sulphoconjugated steroids formed were less than 0·5% of total steroids. Although 19-nortestosterone could not be generated by equine purified aromatase incubated with labelled testosterone, the synthesis of 19-norandrogens and oestrogens by equine placental explants was blocked by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole. Our results provide evidence for a placental origin of at least a part of the 19-norandrogens previously identified in the blood of the pregnant mare. Furthermore, it is suggested that 19-norandrogen biosynthesis would involve the enzymatic metabolism of 19-oxygenated androgens formed by equine aromatase. Journal of Endocrinology (1995) 144, 517–525

Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity

1972 ◽  
Vol 11 (3) ◽  
pp. 855-873
Author(s):  
A. M. LEVINE ◽  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Acyltransferase Activity ◽  
Structural Localization ◽  
Differentiated Cells ◽  
Golgi Membranes

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35% of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.

Studies on the effects of magnesium ion and propranolol on iris muscle phosphatidate phosphohydrolase

1984 ◽  
Vol 62 (2-3) ◽  
pp. 170-177 ◽  
Author(s):  
Ata A. Abdel-Latif ◽  
Jack P. Smith
Keyword(s):  
Smooth Muscle ◽  
Divalent Cation ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Soluble Enzyme ◽  
Microsomal Enzyme ◽  
Time Intervals ◽  
Phosphatidate Phosphohydrolase

The properties, subcellular distribution, and the effects of Mg2+ and propranolol on phosphatidate phosphohydrolase (EC 3.1.3.4) from rabbit iris smooth muscle have been investigated. The particulate and soluble (0–30% (NH4)2SO4 fraction) enzymes were assayed using aqueous phosphatidate dispersions and membrane-bound phosphatidate as substrates, respectively. When measured with aqueous substrate, activity was detected in both the particulate and soluble fractions, with the highest relative specific activity found in the microsomal fraction. Maximum dephosphorylation by the microsomal enzyme was about 1100 nmol of inorganic phosphate released/h per milligram protein and occurred at pH 7.0–7.5. In general Mg2+ inhibited the phosphohydrolase activity of the microsomal fraction and stimulated that of the soluble fraction, and the effects of the divalent cation on both of these activities were reversed by propranolol. The microsomal enzyme was slightly stimulated by deoxycholate and inhibited by the divalent cations Mg2+, Ca2+, and Mn2+ at concentrations > 0.25 mM. In contrast, the soluble enzyme was stimulated by Mg2+. Inhibition of the microsomal enzyme by Mg2+ (0.5 mM) was reversed by both EDTA, which also stimulated at higher concentrations (1 mM), and propranolol (0.1–0.2 mM). The inhibitory effect of Ca2+ on the enzyme was not reversed by propranolol. In the absence of Mg2+, the microsomal enzyme was inhibited by propranolol in a dose-dependent manner, and both in the absence and presence of the divalent cation the soluble enzyme was inhibited by the drug in a similar manner. These data suggest that the cationic moiety of propranolol may act by competing at the Mg2+-binding sites. Addition of propranolol (0.2 mM) to iris muscle prelabelled with [14C]arachidonic acid increased accumulation of [14C]phosphatidic acid at all time intervals (2.5–90 min) and brought about a corresponding initial decrease in the formation of [14C]diacylglycerol at short time intervals (2.5 min), thus implicating the phosphohydrolase as a possible site of action of the drug on glycerolipid metabolism in this tissue. In addition to reporting on the characteristics and distribution of phosphatidate phosphohydrolase in the iris smooth muscle, the data presented add further support to our hypothesis that propranolol redirects glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in their biosynthesis.

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