Transient activation of calcineurin during thiol modification.
Thiol titrations of bovine brain calcineurin, phosphatase with Ellman's reagent revealed the presence of 5 exposed sulfhydryl groups on the native protein, and 10 sulfhydryl groups on the denatured protein. Attempts were made to identify the location of the free thiols within the catalytic and regulatory domains of the enzyme. Our data indicates that free sulfhydryl groups are absent from the vicinity of the Mg2+ and calmodulin binding sites as well as from the active site of the enzyme. However, the fact that the number of free thiols decreased in the presence of Ca2+ and Mn2+, to 4 and 2 respectively, possibly indicates that either free thiols are at or near these domains or become inaccessible as a consequence of conformational changes induced by the metal ions. The Ca2+ and Ca2+/Mg2+ stimulated activities of calcineurin were monitored during modification with Ellman's reagent, iodoacetate and iodoacetamide. Upon modification of 1-2 of the free thiols the activity of the enzyme increased 1.3 to 10.5-fold depending on the thiol reagent and the stimulatory metal ions employed. Modification of the remainder of the free thiols resulted in a decrease in activity. These results suggest that 1-2 thiols are essential for the full expression of calcineurin activity.
Wagner
J
J
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Mutus
B
B
D001665:Binding Sites / Q000502:physiology; D019703:Calcineurin; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D004789:Enzyme Activation / Q000502:physiology; D005978:Glutathione / Q000031:analogs & derivatives; D019803:Glutathione Disulfide; D007460:Iodoacetamide; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008274:Magnesium / Q000502:physiology*; D008345:Manganese / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
D002148:Calmodulin-Binding Proteins; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D008345:Manganese; D004228:Dithionitrobenzoic Acid; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases; D005978:Glutathione; D008274:Magnesium; D002118:Calcium; D019803:Glutathione Disulfide; D019807:Iodoacetic Acid; D007460:Iodoacetamide