Effect of activation of protein phosphatase 1 on sulfhydryl reactivity.
Myofibril protein phosphatase 1 (PP1) from bovine heart, identified as PP1alpha, 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 PP1alpha was associated with 57Co2+ 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'-dithiobis[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 accessible to chemical modification.
Chu
Y
Y
Department of Pharmacology, Medical College of Ohio, Toledo 43699, USA.
Lee
E Y
EY
Reimann
E M
EM
Wilson
S E
SE
Schlender
K K
KK
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003035:Cobalt / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; D003035:Cobalt; D008345:Manganese; D004228:Dithionitrobenzoic Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid; D004229:Dithiothreitol