scholarly journals A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy

1973 ◽  
Vol 134 (1) ◽  
pp. 103-112 ◽  
Author(s):  
E. Heather Mangiapane ◽  
Katherine A. Lloyd-Davies ◽  
David N. Brindley
Keyword(s):  
Partial Hepatectomy ◽  
Microsomal Fraction ◽  
Actinomycin D ◽  
Specific Activity ◽  
Supernatant Fraction ◽  
Glycerol Phosphate ◽  
Liver Remnant ◽  
Control Value ◽  
Phosphatidate Phosphohydrolase ◽  
Subtotal Hepatectomy

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.

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.

ACTIVATION OF SOLUBILIZED STEROID-TRANSFORMING ENZYMES OF ADRENAL MICROSOMAL ORIGIN BY SERUM PROTEINS

1972 ◽  
Vol 54 (2) ◽  
pp. 297-315 ◽  
Author(s):  
MARGOT A. HAMILTON ◽  
R. W. McCUNE ◽  
SIDNEY ROBERTS
Keyword(s):  
High Speed ◽  
Microsomal Fraction ◽  
Adrenal Glands ◽  
Serum Proteins ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fraction ◽  
Rat Serum ◽  
Microsomal Preparation ◽  
Stimulation Of

SUMMARY The reactions which result in the conversion of pregnenolone to progesterone and of progesterone to deoxycorticosterone in undisrupted microsomal preparations from rat adrenal glands were stimulated by homologous serum. The active materials were shown to be firmly associated with serum proteins. The dialysable fraction of serum was either without effect on these transformations or was inhibitory. The enzyme systems involved were partially solubilized by exposure of the microsomal preparation to prolonged sonic treatment or to 1% Triton N-101. After either treatment, 35–40% of the original specific activity of the steroid 21-hydroxylase system responsible for the conversion of progesterone to deoxycorticosterone was found in the supernatant fraction after high-speed centrifugation. However, this solubilized system did not respond to serum preparations. The same procedures also resulted in a supernatant fluid which showed about 50–60% of the initial specific activity of the multi-enzyme system involved in the conversion of pregnenolone to progesterone. In these instances, the stimulatory effect of serum was retained or accentuated. Acetone powders prepared from the adrenal microsomal fraction were also active in the conversion of pregnenolone to progesterone and responded to serum with enhanced activity. Earlier observations that activation of steroid 21-hydroxylase by homologous rat serum was specific for the β-globulin fraction were confirmed in the present investigations. In contrast, stimulation of the conversion of pregnenolone to progesterone was apparently due principally to the albumin fraction. Albumin preparations from a number of other sources, as well as whole human serum protein, were also effective in this regard. The active protein preparations selectively stimulated 4-ene-3β-hydroxysteroid dehydrogenase activity, but did not activate 5-ene-3-oxosteroid isomerase in the microsomal fraction. This finding suggested that activation of 5-ene-3β-hydroxysteroid dehydrogenase was responsible for stimulation of progesterone synthesis from pregnenolone. The results indicate that the protein-bound factor in rat serum which was capable of stimulating the conversion of progesterone to deoxycorticosterone in microsomal preparations from rat adrenal glands was different from that which activates the conversion of pregnenolone to progesterone. Moreover, these diverse factors appeared to act by different mechanisms.

scholarly journals The effect of starvation on the incorporation of palmitate into glycerides and phospholipids of rat liver homogenates

1969 ◽  
Vol 115 (2) ◽  
pp. 139-145 ◽  
Author(s):  
M. Vavrečka ◽  
M P Mitchell ◽  
G. Hübscher
Keyword(s):  
Specific Activity ◽  
Liver Homogenate ◽  
Glycerol Phosphate ◽  
Total Liver ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound ◽  
Total Dna ◽  
Liver Homogenates ◽  
Rate Limiting ◽  
Increased Activity

1. Glyceride biosynthesis from glycerol phosphate and [1−14C]palmitate was studied in liver homogenates of rats that were fed ad libitum or starved for 36–40hr. The changes in enzyme activity were related to total DNA content or total liver homogenate as these were found to be equivalent and to be the most meaningful parameters. 2. In liver homogenates from fed rats, labelled palmitate was incorporated mainly into phosphatidate (58% of the total incorporation into lipids), diglycerides (25%) and triglycerides (16%), whereas monoglycerides, cholesterol esters and phospholipids other than phosphatidate were labelled only to a small extent. Addition of particle-free supernatant to full homogenates increased the total incorporation of palmitate by 45% and the pattern of incorporation altered to 53% incorporated into triglycerides, 24% into diglycerides and 17% into phosphatidate. This result suggested that, in liver homogenates, phosphatidate phosphohydrolase (EC 3.1.3.4) may be rate-limiting in the biosynthesis of glycerides via the glycerol phosphate pathway. 3. Upon starvation, the amount of palmitate incorporated per liver into total phospholipids plus glycerides was decreased to between 68% and 75% of that observed with fed animals. In homogenates from fed animals 41–44% of the labelled phospholipids plus glycerides was in glycerides; this value increased to between 63% and 75% with starved rats. Of the palmitate incorporated into total phospholipids, between 85% and 86% was found in phosphatidate, independent of the nutritional state of the animal. The ratio of palmitate incorporated into triglycerides/diglycerides rose from 0·7, obtained with fed rats, to 1·0 with starved animals. 4. These results indicate that starvation caused a decrease in the activity (per total liver) of acyl-CoA–glycerol phosphate acyltransferase(s) (EC 2.3.1.15) and an increase in the activity of acyl-CoA–diglyceride acyltransferase (EC 2.3.1.20). The largest change, however, seemed to be related to the increased activity of the phosphatidate phosphohydrolase in the particle-free supernatant. 5. The latter enzyme was assayed in the particle-free supernatant with membrane-bound phosphatidate as substrate. In starvation, the activity per total liver was increased to between 130% and 190% and the specific activity to between 180% and 320% of the values for fed rats.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

scholarly journals Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

1968 ◽  
Vol 109 (4) ◽  
pp. 485-494 ◽  
Author(s):  
N. M. Wilkie ◽  
R. M. S. Smellie
Keyword(s):  
Rat Liver ◽  
Alkaline Hydrolysis ◽  
Actinomycin D ◽  
Chain Extension ◽  
Supernatant Fraction ◽  
Inorganic Pyrophosphate ◽  
Optimum Ph ◽  
Microsome Fraction ◽  
Hydrolysis Of ◽  
Generating System

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg2+ ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2–3 uridine residues, and there is evidence that these may be added to the 3′-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg2+ ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5′-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5′-triphosphate. The optimum pH is in the range 7·0–8·5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [32P]UTP in the absence of ATP, GTP and CTP, 80–90% of 32P was recovered in UMP-2′ or −3′ after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the 32P was recovered in nucleotides other than UMP-2′ or −3′. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.

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

METABOLISM OF PROGESTERONE BY THE RAT OVARY: FORMATION OF 3β-HYDROXY-5α-PREGNAN-20-ONE BY OVARIAN MICROSOMES

1969 ◽  
Vol 61 (4) ◽  
pp. 618-628 ◽  
Author(s):  
A. Zmigrod ◽  
H. R. Lindner
Keyword(s):  
Oxidation Product ◽  
Microsomal Fraction ◽  
Biological Function ◽  
Specific Activity ◽  
Metabolite Identification ◽  
Enzymic Activity ◽  
Chromatographic Behaviour ◽  
Rat Ovary ◽  
Chromatographic Characterization

ABSTRACT A microsomal fraction from rat ovaries incubated with [4-14C] progesterone in the presence of NADPH produced radioactive 3β-hydroxy-5α-pregnan-20-one as the major metabolite. Identification of this compound, not hitherto isolated from mammalian ovaries, was based on (i) paper and gas-chromatographic behaviour of the free compound and its acetate, (ii) gas-chromatographic characterization of the thioketal derivative and the oxidation product, and (iii) recrystallization with authentic carrier to constant specific activity. Ovaries from both pregnant and prooestrous rats showed this enzymic activity. The possible biological function of ovarian steroid reductases is discussed.

Biosynthesis of Nitrogenous Phospholipids in Spinach Leaves

1974 ◽  
Vol 52 (6) ◽  
pp. 469-482 ◽  
Author(s):  
M. O. Marshall ◽  
M. Kates
Keyword(s):  
Microsomal Fraction ◽  
Enzyme System ◽  
Supernatant Fraction ◽  
Exchange Reactions ◽  
Methylation Pathway ◽  
Ethanolamine Kinase ◽  
Spinach Leaves

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

Biosynthesis of 1,2-3H and 4-14C steroid sulfates of high specific activity

1970 ◽  
Vol 48 (1) ◽  
pp. 148-150 ◽  
Author(s):  
J. Torday ◽  
G. Hall ◽  
M. Schweitzer ◽  
C. J. P. Giroud
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Homogenate ◽  
High Specific Activity ◽  
Supernatant Fraction ◽  
Metabolic Studies

A supernatant fraction of rat liver homogenate enriched with ATP was used for the biosynthesis of the ester sulfates of several 3H and 14C steroids of the pregn-4-ene series. The method provides a simple means to prepare steroid sulfates of high specific activity for use in either metabolic studies or as reference compounds in the quantification of such conjugates by isotope assays.

scholarly journals Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes

1985 ◽  
Vol 230 (2) ◽  
pp. 525-534 ◽  
Author(s):  
R A Pittner ◽  
R Fears ◽  
D N Brindley
Keyword(s):  
Neutral Lipids ◽  
Relative Proportion ◽  
Rat Hepatocytes ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Phosphatidate Phosphohydrolase ◽  
Cultured Rat Hepatocytes ◽  
Total Glycerol

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.

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