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Studies of rat adipose-tissue microsomal glycerol phosphate acyltransferase.

ABSTRACT: Incubation of rat adipose-tissue microsomal fractions with iodoacetate caused an inactivation of glycerol phosphate acyltransferase that could be prevented by the presence of palmitoyl-CoA. A microsomal protein of subunit Mr 54 000 was found to react with radioactively labelled iodoacetate in the absence, but not in the presence, of palmitoyl-CoA. It is suggested that this protein is a component of glycerol phosphate acyltransferase. Incubation of rat adipose-tissue microsomal fractions with the catalytic subunit of cyclic AMP-dependent protein kinase, ATP and Mg2+ caused an inactivation of glycerol phosphate acyltransferase whose magnitude depended on the conditions used for assay of the acyltransferase. Rat adipose tissue microsomal proteins were phosphorylated by using protein kinase and [gamma-32P]ATP. One of the phosphorylated proteins was very similar, but not identical, in mobility to the Mr-54 000 protein labelled by iodoacetate. In contrast with a previous report [Sooranna & Saggerson (1976) FEBS Lett. 64, 36-39], no changes could be detected in the activity of glycerol phosphate acyltransferase in adipocytes treated with adrenaline. Adipocytes were labelled with [32P]Pi and treated with adrenaline, but no 32P was incorporated into the Mr-54000 protein labelled by iodoacetate. The results suggest that the activity of adipose-tissue microsomal glycerol phosphate acyltransferase is not directly controlled by phosphorylation.

SUBMITTER: Nimmo GA

PROVIDER: S-EPMC1144402 | biostudies-other | 1984 Nov

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.

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Studies of rat adipose-tissue microsomal glycerol phosphate acyltransferase.

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