|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1990 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Phosphorylation-dephosphorylation of myosin plays as important role in the regulation of Smooth muscle activity. Myosin light chain kinase is well characterized, but less is known about the phosphatase involved. Thus, the purification, characterization and cDNA cloning of smooth muscle myosin light chain phosphatase were done. We purified the endogeneous phosphatase of smooth muscle from the crude actomyosin of chicken gizzard by chromatography on DEAE-cellulofine, heparin-S heparin-Sepharose and a thiophosphorylated myosin light chain-Sepharose affinity column. The specific activity of this phosphatase, purified about 1500 fold, was 75 nmol Pi/min /mg. The purified phophatase contained two subunits of Mr 58 and 38 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 38 kDa peptide is a catalytic subunit of a phosphatase and the 58 kDa one is thought to be a regulatory subunit having myosin binding property. This preparation was active with both isolated light chains a
nd intact myosin phosphorylated by myosin light chain kinase, suggesting that this phosphatase is a myosin light chain phtsphatase (MLCP). Purified MLCP was identified as PP-1delta by Western blotting using polyclonal antibodies directed against the distinct C-terminal peptides of PP-1alpha, PP-1delta and PP-2A catalytic subunits. A cDNA clone containing the sequence of a PP-1delta catalytic subunit has been isolated from a chichen gizzard lambdagt11 library by using the cDNA fragment of PP-1 amplified by RT-PCR method from rat liver as a probe. The insert size of this clone was 1.3 kb. The partial sequence analyses of the insert revealed that it has the nucleotide identity of more than 80% to rat liver PP-1delta. These results suggest that smooth muscle myosin light chain phosphatase is a myosin-bound type PP-1delta and its catalytic subunit has high homology with rat liver PP-1delta.