Characterization of insulin-sensitive phosphodiesterase in fat cells. I. Effects of salts and oxidation-reduction agents.

Abstract

Several basic characteristics of insulin-sensitive "lowKm" phosphodiesterase were studied in a crude microsomal fraction of rat epididymal fat cells.At 37"C, the enzyme activities (both basal and plus insulin) increased in a time-dependent manner in a high salt solution, remained almost unchanged in 0.1 M KC1 or NaCl, and decreased in a low salt medium.In the presence of 0.1 M KC1 at 37OC, the enzyme activities were suppressed by dithiothreitol, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, but not by H202 or iodoacetamide, all at 1 m ~.The inhibitory effect of dithiothreitol was reversed upon subsequent incubation with HzOz.When the enzyme was incubated with dithiothreitol for 24 h at 4"C, the basal activity increased to the plus insulin level, the plus insulin activity remained unchanged, and both activities became water-soluble.The Stokes radii of the preparations solubilized from the basal and plus insulin enzymes were both approximately 53 A. This value was almost equal to that of the active component solubilized by a mild trypsin treatment.By a mild trypsin treatment, the phosphodiesterase preparations that had been deactivated by low salt or dithiothreitol treatment at 37°C (see above) were both fully reactivated.In contrast, the enzyme inactivated with pchloromercuriphenylsulfonate was not reactivated.