Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular system from rat adipocytes. II. Characterization of insulin-sensitive phosphoproteins and conditions for observation of the insulin effect

Seals, J.R.; McDonald, J.M.; Jarett, L.

Journal of Biological Chemistry 254(15): 6997-7001


ISSN/ISBN: 0021-9258
PMID: 222753
Accession: 068524652

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The direct addition of insulin to a plasma membrane preparation from rat adipocytes specifically decreased the phosphorylation of a plasma membrane protein of 120,000 daltons (band 13) and a protein of 42,000 daltons whose origin is in mitochondria contaminating the plasma membrane preparation (band 6). Analysis of these insulin-sensitive bands revealed them both to be phosphoesters, primarily phosphoserine. However, the responses of bands 6 and 13 to variations of pH, ionic composition and the presence of cAMP or various chemical agents in the phosphorylation medium indicated that 2 distinct systems were involved in the phosphorylation and the insulin effect on these 2 bands. Insulin effects can be observed in a subcellular system consisting of plasma membranes and mitochondria. The insulin effect on band 13 phosphorylation results from the interaction of insulin with its plasma membrane receptor. The insulin effect on mitochondrial contaminant band 6 appears to be secondary to interaction of the hormone with the plasma membrane. Insulin had no direct effect on the phosphorylation of band 6 when added to mitochondria alone. However, when insulin was added to a mixture of plasma membranes and added mitochondria, the effect was reconstituted. Band 6 appears to be the .alpha. subunit of pyruvate dehydrogenase, an insulin-sensitive enzyme, the phosphorylation of which regulates the activity of the enzyme. In the subcellular system used, insulin is capable of interacting with a specific plasma membrane receptor and directly activates its effector system, leading to the generation of a second messenger which brings about the observed changes in mitochondrial band 6 and possibly in plasma membrane band 13.