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A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

ABSTRACT: Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

SUBMITTER: Rider MH

PROVIDER: S-EPMC1152233 | biostudies-other | 1983 Jul

REPOSITORIES: biostudies-other

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Project description:Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.

Project description:1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.

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A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

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