Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1 alpha, was purified in a latent form which was dependent on Co2+ or Mn2+ for activity (Y. Chu, S. E. Wilson, and K. K. Schlender (1994) Biochim. Biophys. Acta 1208, 45-54). This was also true for recombinant PP1 alpha expressed in Escherichia coli (Z. Zhang, G. Bai, S. Deans-Zirattu, M. F. Browner, and E. Y. C. Lee (1992) J. Biol. Chem. 267, 1484-1490). Here we report on the change in the sulfhydryl reactivity during the cation activation process. The activation of myofibrillar PP1 by Co2(+) was prevented by 10 mM dithiothreitol (DTT) and incubation of the Co2+-activated enzyme with 50 mM DTT reversed the activation. Activation of recombinant PP1 alpha was associated with Co-57(2+) incorporation into PP1. DTT reversal of Co2+-activated PP1 was accompanied by release of Co2+ from the enzyme. The latent PP1 modified with 2-nitro-5-thiocyanobenzoic acid (NTCB) or N-ethylmaleimide (NEM) did not bind Co2+ and could not be activated by Co2+. Conversely, the Co2+-activated PP1 was resistant to inactivation with NTCB and less sensitive to NEM. Similarly, PP1 pretreated with NTCB was not activated by Mn2+ and the Mn2+-activated enzyme was also resistant to NTCB inhibition. The number of sulfhydryls of nondenatured PP1, reactive with 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB), was reduced from approximately 8 to 2-3 mol/mol when the enzyme was activated with Co2+ or Mn2+. After denaturation with guanidine-HCl, the number of reactive sulfhydryls of nonactivated PP1 and Co2+-activated PP1 was approximately 10 mol/mol enzyme. These results suggest that when PP1 is activated by Co2+ or Mn2+, the enzyme undergoes a conformational change resulting in some of the cysteine sulfhydryls no longer being accessable to chemical modification. (C) 1996 Academic Press, Inc.
