ACTIVATION OF ENZYMES BY CALMODULINS CONTAINING INTRAMOLECULAR CROSS-LINKS

We have reacted calmodulins containing cysteines substituted at positi ons 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate i ntramolecularly cross-linked proteins termed BMHCM or BMHCM1, respecti vely. Reactions were also performed with N-ethylmaleimide (NEM) in pla ce of BMH to generate corresponding S-ethylsuccinimidylated proteins t ermed NEMCM or NEMCM1. The abilities of these proteins to activate pla nt NAD kinase, erythrocyte Ca2+-ATPase and bovine brain calcineurin ac tivities were assessed. The BMH- or NEM-reacted proteins activate calc ineurin activity as does control calmodulin. K(act) values for Ca2+-AT Pase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in V(max) values. Acti vation of this enzyme by NEMCM or NEMCM1 is not different from the con trol. In NAD kinase activation experiments BMHCM and BMHCM1 are associ ated with a 10 to 20-fold increase in K(act) values and a 60-75% reduc tion in V(max) values relative to the control. NEMCM1 is not associate d with any apparent changes in NAD kinase activation, however, NEMCM i s associated with a 10-fold increase in the K(act) value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased K(act) value, implying that this change is due to in teractions between S-(ethylsuccinimido)cysteines at positions 3 and 14 6. In conclusion, cross-linking and associated distortions in the stru cture of calmodulin appear to have little or no effect on activation o f calcineurin enzyme activity. However, bending in the central helix a nd/or steric restrictions associated with cross-linking increase signi ficantly the K(act) value for Ca2+-ATPase and NAD kinase activation, a nd dramatically reduce maximal activation of NAD kinase activity.