INTERACTIONS AND PROPERTIES OF SMOOTH-MUSCLE MYOSIN PHOSPHATASE

Interactions of the type 1 phosphatase catalytic subunit (PP1c) and th e myosin phosphatase holoenzyme (MBP) were compared using affinity col umns. In the absence of ATP, MBP bound to dephosphorylated myosin, hea vy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not boun d. In the presence of ATP, the binding of MBP occurred only with phosp horylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitiv e inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K-m for the latter. With myosin, MBP showed a marked increase in k(cat), compared to PP1c, ATP did not affect these parameters. To i nvestigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were e xpressed. These activated PP1c, and activation was maximum at approxim ately an equimolar ratio. The equimolar mixture of recombinant fragmen t and PP1c exhibited K, values similar to MBP and increased k,,, value s, compared to PP1c alone. An affinity column was prepared using the r ecombinant fragment. Phosphorylated HMM and P-LC20 were bound in the p resence and absence of ATP. The interaction of P-LC20 was not ATP-depe ndent. Dephosphorylated HMM did not bind in the presence of ATP. The N -terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the la rge subunit of MBP contained binding sites for P-LC20 and PP1c.