Factors responsible for the Ca(2+)-dependent inactivation of calcineurin in brain

• Published in: FEBS letters 1995-10, Vol.374 (2), p.237
• Main Authors: Stemmer, P M, Wang, X, Krinks, M H, Klee, C B
• Format: Article
• Language: English
• Subjects: Animals; Brain - enzymology; Calcineurin; Calcium - pharmacology; Calmodulin - pharmacology; Calmodulin-Binding Proteins - antagonists & inhibitors; Calmodulin-Binding Proteins - metabolism; Enzyme Inhibitors - pharmacology; Ethers, Cyclic - pharmacology; Immunosuppressive Agents - pharmacology; Male; Okadaic Acid; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoprotein Phosphatases - metabolism; Polyenes - pharmacology; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus - analogs & derivatives; Tacrolimus - pharmacology; Tissue Extracts
• ISSN: 0014-5793
• DOI: 10.1016/0014-5793(95)01095-V

The Ca(2+)-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimulated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca2+ and inhibited by the calmodulin-binding peptide, M13, and by the immunosuppressive drug, FK506. It is insensitive to rapamycin at concentrations up to 1 microM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).