Cyclic nucleotide phosphodiesterase activities from isolated fat cells: Correlation of subcellular distribution with effects of nucleotides and insulin

doi:10.1016/0003-9861(74)90190-8

Archives of Biochemistry and Biophysics

Volume 162, Issue 2, June 1974, Pages 331-339

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Abstract

Cyclic nucleotide phosphodiesterasc activities were determined in fractions of fat cell homogenates, prepared either by differential centrifugation or by centrifugation on discontinuous sucrose gradients.

In the supernatant fraction (150,000g supernatant in 0.25 m sucrose, or 92,000g supernatant in 0.32m sucrose): (a) there was 70% of the cyclic AMP phosphodiesterase activity of the whole homogenate, and over 90% of the cyclic GMP phosphodiesterase activity; (b) double reciprocal kinetic plots were nonlinear for both substrates; (c) cyclic (GMP, 0.02-2 μm, activated hydrolysis of 10 μm cyclic AMP; (d) 25 or 50 μm cyclic GMP noncompetitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 38 μm); (e) cyclic AMP, 0.1 μm, slightly activated hydrolysis of 10 μm cyclic GMP; (f) 10 or 20 μm cyclic AMP competitively inhibited hydrolysis of 5–20 μm cyclic GMP (K_i = 18 μm).

In the particle fraction (1000g, 1000-16,000g, and 16,000–150,000g pellets in 0.25m sucrose, or 0.8-1.2m sucrose interface at 92,000g): (a) there was 30% of the cyclic AMP phosphodiesterase activity of whole homogenate, but less than 5% of the cyclic GMP phosphodiesterase; (b) the double reciprocal kinetic plot of hydrolysis of cyclic AMP was nonlinear; (c) cyclic GMP, 0.02-2μm, did not affect hydrolysis of 10 μm cyclic AMP; (d) 5 or 10 μm cyclic GMP competitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 1.9 μm).

Incubation of fat cells with insulin, 40 ng/ml, increased the maximum velocity of particulate high-affinity cyclic AMP phosphodiesterase, but did not affect the supernatant activity. Addition of insulin after homogenization of the cells had no effect on any phosphodiestesterase activity.

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^☆: This work was supported by grants AM 15263, AM 05020, and AM 02456 from the U.S. Public Health Service, NIAMDD, and by a research grant to V. R. Lavis from the American Diabetes Association. W. J. Thompson was supported by postdoctoral fellowship 1 FO2 AM 50789 from the U.S. Public Health Service, NIAMDD.

²: Present address: Nagoya City University, School of Medicine, Department of Biochemistry, Nagoya, Japan.

³: Present address: University of Texas Medical School at, Houston, Program in Pharmacology, Houston, Texas.

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Cyclic nucleotide phosphodiesterase activities from isolated fat cells: Correlation of subcellular distribution with effects of nucleotides and insulin☆

Abstract

J. Biol. Chem

Life Sci

Arch. Biochem. Biophys

Arch. Biochem. Biophys

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem

Biochim. Biophys. Acta

Advan. Enzyme Regul

J. Biol. Chem

Life Sci

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun

Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol

Biochem. J

J. Lab. Clin. Med