| Abstrakt: |
Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1α, 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. Acta1208, 45–54). This was also true for recombinant PP1α expressed inEscherichia 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 mmdithiothreitol (DTT) and incubation of the Co2+-activated enzyme with 50 mmDTT reversed the activation. Activation of recombinant PP1α was associated with57Co2+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) orN-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. |
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