Factors responsible for the Ca(2+)-dependent inactivation of calcineurin in brain.
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).
Stemmer
P M
PM
Laboratory of Biochemistry, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Wang
X
X
Krinks
M H
MH
Klee
C B
CB
D000818:Animals; D001921:Brain / Q000201:enzymology*; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008297:Male; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011090:Polyenes / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020123:Sirolimus; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D014020:Tissue Extracts
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D007166:Immunosuppressive Agents; D011090:Polyenes; D014020:Tissue Extracts; D019319:Okadaic Acid; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium; D020123:Sirolimus; D016559:Tacrolimus