Purification and characterization of a smooth muscle myosin light chain kinase-phosphatase complex.
We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SP-Sepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase- and myosin-associated protein phosphatase (KAMPPase). The KAMPPase multienzyme complex was composed of a 37-kDa catalytic (PC) subunit, a 67-kDa targeting (PT) subunit, and MLCKase with or without CaM. The approximate molar ratio of the PC and PT subunits was 1:2 with a variable and usually higher molar content of MLCKase. The targeting role of the PT subunit was directly demonstrated in binding experiments in which the PT subunit bound to both the kinase and to CaM. Its binding to CaM was, however, Ca2+-independent. MLCKase and the PT subunit potentiated activity of the PC subunit when intact myosin was used as the substrate. These data indicated that there is a Ca2+-independent interaction among the MLCPase, MLCKase, and CaM that are involved in the regulation of phosphatase activity.
Sobieszek
A
A
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria. ASobieszek@oeaw.ac.at
Borkowski
J
J
Babiychuk
V S
VS
D000818:Animals; D001717:Birds; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000302:isolation & purification* / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism
D009097:Multienzyme Complexes; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase