FACTORS RESPONSIBLE FOR THE CA2(-DEPENDENT INACTIVATION OF CALCINEURIN IN BRAIN())

The Ca2+-dependent protein phosphatase activity of crude rat brain ext racts measured in the presence of okadaic acid, exhibits the character istic properties of the calmodulin-stimulated protein phosphatase, cal cineurin. It is stimulated more than 200-fold by Ca2+ and inhibited by the calmodulin-binding peptide, M13, and by the immunosuppressive dru g, PK506. It is insensitive to rapamycin at concentrations up to 1 mu M. Its specific activity, based on calcineurin concentration determine d by quantitative analysis of Western blots exposed to anti-bovine bra in IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions, Unlike the purified enzyme it is rap idly and irreversibly inactivated in a time-, temperature-, and Ca2+/c almodulin-dependent fashion without evidence of extensive proteolytic degradation, The enzyme is converted to a state which does not lose ac tivity by removal of low molecular weight material by gel filtration, Reconstitution of a labile enzyme is achieved by the addition of the l ow molecular weight-containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain e xtracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).