Insulin-sensitive phosphodiesterase. Its localization, hormonal stimulation, and oxidative stabilization

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Insulin stimulates membrane-bound phosphodiesterase (cyclic adenosine 3':5'-monophosphate phosphodiesterase) in rat epididymal fat cells.
• Previous studies indicated insulin's stimulatory effect on this enzyme.

Purpose of the Study:

• To investigate the subcellular localization of insulin-sensitive phosphodiesterase.
• To elucidate the role of sulfhydryl groups and oxidation in enzyme activity regulation.
• To understand the influence of metal ions and reducing/oxidizing agents on enzyme function.

Main Methods:

• Sucrose density gradient centrifugation of microsomal fractions.
• Homogenization of fat cells in the presence of various agents (inhibitors, reducing agents, chelators, oxidants).
• Enzyme activity assays for phosphodiesterase, 5'-AMPase, adenylate cyclase, and oxidoreductases.
• Partial purification and stability assessment of the enzyme.

Main Results:

• Insulin-sensitive phosphodiesterase activity was primarily localized in the "light" microsomal fraction, distinct from "heavy" microsomal markers.
• Enzyme activity was diminished by sulfhydryl-blocking agents and reducing conditions, but enhanced by oxidants.
• The activity of the oxidized enzyme could be reversed by reducing agents.
• Metal-chelating agents like EGTA inhibited basal and norepinephrine-stimulated activity, an effect reversible by heavy metals but not Ca2+ or Mg2+.
• The enzyme exhibited stability in both basal and stimulated states after partial purification.

Conclusions:

• Insulin-sensitive phosphodiesterase and established cell membrane markers are associated with different subcellular components.
• The insulin-stimulated phosphodiesterase likely possesses critical sulfhydryl groups whose oxidation stabilizes enzyme activity.
• Heavy metal ions may catalyze the air oxidation of the enzyme, a process inhibitable by metal-chelating agents.