Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle.
Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.
Erdödi
F
F
Department of Biological Chemistry, College of Medicine, University ofIllinois Chicago.
Rokolya
A
A
Bárány
M
M
Bárány
K
K
D000818:Animals; D001011:Aorta / Q000201:enzymology; D002352:Carrier Proteins; D002845:Chromatography; D002850:Chromatography, Gel; D006493:Heparin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000494:pharmacology; D013552:Swine; D000097847:Protein Phosphatase Inhibitory Proteins
D002352:Carrier Proteins; D006493:Heparin; D047908:Intracellular Signaling Peptides and Proteins; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins; D010749:Phosphoprotein Phosphatases; D010764:Phosphorylase Kinase; D011506:Proteins; C021042:protein phosphatase inhibitor-1; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins