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.

Relative suitability of 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glycerylphosphorylcholine and different acyl donors for phosphatidylcholine synthesis via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes

1980 ◽  
Vol 58 (5) ◽  
pp. 434-439 ◽  
Author(s):  
B. J. Holub ◽  
J. Plekarski ◽  
F. Possmayer
Keyword(s):  
Fatty Acids ◽  
The Other ◽  
Acyl Donor ◽  
Rat Lung ◽  
Acceptor Reaction ◽  
Acyl Acceptor ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Acyl Donors ◽  
Phosphatidylcholine Synthesis ◽  
Assay Conditions

The relative suitability of the 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glyceryl-phosphorylcholine and different acyl donors were tested as substrates for phosphatidylcholine synthesis via the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes. The acyl acceptor was an almost equi-molar mixture of the [3H]palmitoyl plus [14C]stearoyl species of 1-acyl-sn-glycero-3-phosphorylcholine with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, or arachidonoyl-CoA serving as the acyl donor. At all concentrations of acyl acceptor, reaction velocities with 20:4-CoA ≥ 18:2-CoA > 18:1-CoA > 16:0-CoA > 18:0-CoA. Furthermore, the acyltransferase selectively utilized the 1-palmitoyl over the 1-stearoyl species of 1-acylglycerylphosphorylcholine by 4.2- to 5.7-fold under optimal assay conditions with the various acyl-CoA thiolesters. However, the degree of preference exhibited for the 1-palmitoyl-sn-glycero-3-phosphorylcholine, as acyl acceptor, versus the 1-stearoyl homologue with palmitoyl-CoA as the acyl donor was not significantly different from that obtained with the other acyl-CoA derivatives. Thus, the specificity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase indicates an ability of this enzyme to produce dipalmitoyl phosphatidylcholine but it cannot independently explain the predominance of dipalmitoyl phosphatidylcholine in lung or the tendency of stearate at the 1-position to associate with fatty acids of increasing unsaturation at the 2-position.

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.

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.

scholarly journals A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture

1988 ◽  
Vol 253 (2) ◽  
pp. 451-457 ◽  
Author(s):  
P A Caesar ◽  
S J Wilson ◽  
C S Normand ◽  
A D Postle
Keyword(s):  
Organotypic Culture ◽  
Fetal Lung ◽  
Reversed Phase ◽  
Lung Cells ◽  
Individual Molecular Species ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Choline Incorporation ◽  
Fractional Synthesis ◽  
Human Fetal Lung ◽  
Phosphatidylcholine Synthesis

Human fetal lung (14-18 weeks gestation) was maintained in either organ or organotypic culture. By 4 days in organ culture or 14 days in organotypic culture, epithelial cells within both culture systems exhibited well-developed apical microvilli and possessed numerous intracellular lamellar bodies characteristic of surfactant phospholipid stores. However, analysis of the pattern of synthesis of individual molecular species of phosphatidylcholine by [14C]choline incorporation and reversed-phase h.p.l.c. showed that this apparent maturation was not paralleled by an increased synthesis of the dipalmitoyl species in either culture system. By contrast, the fractional synthesis of dipalmitoyl phosphatidylcholine, expressed as a percentage of total [14C]choline incorporation, decreased with time in both organ and organotypic culture. Moreover, these fractions were not significantly different from those measured in parallel monolayer cultures of mixed human fetal lung cells that displayed mainly fibroblast morphology. These results suggest that the synthesis pattern of phosphatidylcholine species by lung cells in culture is determined principally by their incubation conditions and not by their state of apparent maturation.

Unsaturated fatty acids in the postnatally developing rat lung

Lipids ◽  
1983 ◽  
Vol 18 (1) ◽  
pp. 50-54
Author(s):  
James P. Kehrer ◽  
Anne P. Autor
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Rat Lung ◽  
Developing Rat

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

THE INCORPORATION OF PALMITATE-1-14C BY RAT LUNG IN VITRO

1967 ◽  
Vol 45 (4) ◽  
pp. 597-607 ◽  
Author(s):  
A. Naimark ◽  
D. Klass
Keyword(s):  
Specific Activity ◽  
Specific Radioactivity ◽  
Phosphatidyl Inositol ◽  
Phosphatidyl Serine ◽  
Rat Lung ◽  
Exchange Reactions ◽  
Ethanolamine Phosphatidyl ◽  
Exogenous Glucose ◽  
Lipid Fractions

The incorporation of palmitate-1-14C into various lipid fractions was studied in rat lung in vitro. Most of the radioactivity was found in phospholipid, although in terms of decreasing specific activity the lipids were ranked: free fatty acid (FFA), glycerides, phospholipid. In addition to the usual glycerophosphatides, rat lung contained a substance with some of the chromatographic characteristics of phosphatidyl dimethylethanolamine. In terms of decreasing specific activities the phospholipids were ranked: phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl dimethylethanolamine, phosphatidyl serine plus phosphatidyl inositol, sphingomyelin plus lysophosphatidyl choline. Inhibition of oxidative energy production by hypoxia, cyanide, or low temperature markedly depressed the esterification of palmitate-1-14C. Less marked depression was observed in the absence of exogenous glucose. In all cases the decreased incorporation was associated with an increase in the total and specific radioactivity in tissue FFA. It is concluded that energy-independent exchange reactions are probably of little importance in the incorporation of FFA into esterified lipids of lung tissue. Under conditions of metabolic inhibition the penetration of labelled FFA into the tissue FFA pool does not appear to limit esterification.

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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