Purification and characterization of a kinase-associated, myofibrillar smooth muscle myosin light chain phosphatase possessing a calmodulin-targeting subunit.
A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase- and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca2+-independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10-20-fold after preincubation with 5-50 microM of CoCl2; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (Ki = 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 microM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.
Sobieszek
A
A
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria. ASobieszek@oeaw.ac.at
Babiychuk
E B
EB
Ortner
B
B
Borkowski
J
J
D000818:Animals; D001665:Binding Sites; D001717:Birds; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase