Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.
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ABSTRACT:
SUBMITTER: Butterwith SC
PROVIDER: S-EPMC1153702 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
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Project description:When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.
| S-EPMC1133467 | biostudies-other
Project description:Lipin 1 is a bifunctional intracellular protein that regulates fatty acid metabolism in the nucleus via interactions with DNA-bound transcription factors and at the endoplasmic reticulum as a phosphatidic acid phosphohydrolase enzyme (PAP-1) to catalyze the penultimate step in triglyceride synthesis. However, livers of 8-day-old mice lacking lipin 1 (fld mice) exhibited normal PAP-1 activity and a 20-fold increase in triglyceride levels. We sought to further analyze the hepatic lipid profile of these mice by electrospray ionization mass spectrometry. Surprisingly, hepatic content of phosphatidate, the substrate of PAP-1 enzymes, was markedly diminished in fld mice. Similarly, other phospholipids derived from phosphatidate, phosphatidylglycerol and cardiolipin, were also depleted. Another member of the lipin family (lipin 2) is enriched in liver, and hepatic lipin 2 protein content was markedly increased by lipin 1 deficiency, food deprivation, and obesity, often independent of changes in steady-state mRNA levels. Importantly, RNAi against lipin 2 markedly reduced PAP-1 activity in hepatocytes from both wild type and fld mice and suppressed triglyceride synthesis under conditions of high fatty acid availability. Collectively, these data suggest that lipin 2 plays an important role as a hepatic PAP-1 enzyme.
| S-EPMC2652272 | biostudies-literature
Project description:Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5'-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.
| S-EPMC1137057 | biostudies-other
Project description:N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.
| S-EPMC1136819 | biostudies-other
Project description:The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline [Cheng & Saggerson (1978) FEBS Lett. 87, 65--68; Cheng & Saggerson (1978) FEBS Lett. 93, 120--124] persists for at least 40 min in crude defatted homogenates kept at 0 degrees C or 20 degrees C, but is diminished at 37 degrees C. The effect of noradrenaline persists through the isolation of post-105000 g supernatants and is then stable in these preparations at 0 degrees C and 37 degrees C. Inclusion of albumin (10--20 mg/ml) in homogenization buffers abolishes the effect of noradrenaline. The effect of noradrenaline is not removed by dialysis of extracts or by raising the concentrations of Mg2+ or phosphatidate in assays.
| S-EPMC1162144 | biostudies-other
Project description:The mitochondrial enzyme phosphate-dependent glutaminase was partially purified from rat liver. The enzyme had Mr 290 000 as judged by chromatography on Sephacryl S-300. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the preparation, glutaminase was tentatively identified with a peptide of Mr 73 500. The concentration-dependence on glutamine was highly sigmoidal, with half-maximum velocity at 22 mM-glutamine. Half-maximum activity was obtained with 5 mM-phosphate. The enzyme required ammonia as an obligatory activator, in agreement with previous reports on intact and sonicated mitochondria. These findings further differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney and several other tissues.
| S-EPMC1153663 | biostudies-other
Project description:Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.
| S-EPMC1186145 | biostudies-other
Project description:An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.
| S-EPMC1184170 | biostudies-other
Project description:Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).
| S-EPMC1151513 | biostudies-other
Project description:Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.
| S-EPMC1149861 | biostudies-other