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Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards

Date

1995-01-01

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Degree Level

Doctoral

Abstract

The contraction and relaxation of smooth muscle depends on the reversible phosphorylation of the 20,000 Da myosin light chains (MLC20). The phosphorylation reaction, which is catalyzed by myosin light chain kinase and results in contraction, has been well characterized. In contrast, little is known about the dephosphorylation reaction or the myosin phosphatase that catalyzes this reaction. The main objective of this project was to study the enzyme that catalyzes the dephosphorylation reaction. To this end a myosin phosphatase termed smooth muscle phosphatase (SMP)-III was purified from turkey gizzards. SMP-III is highly specific for myosin and MLC20. The limited proteolysis of SMP-III results in a 1.5- and 2.3-fold increase in the Vmax for MLC20 and HMM (heavy meromyosin), respectively. The increase in activity correlates with the loss of a peptide of approximately 1 kDa from the C terminus. Purified SMP-III has a molecular weight of 390,000 Da as determined by gel filtration chromatography and a catalytic subunit of 38,000 Da as determined from non-denaturing polyacrylamide gel electrophoresis and Western blot analysis. Two proteins of 130 and 22 kDa co-purify with the SMP-III catalytic subunit and may be regulatory subunits of the phosphatase. SMP-III has structural similarities with type 1 protein phosphatases as determined by immunocross-reactivity and proteolytic peptide amino acid sequencing. However, as isolated, SMP-III is enzymatically distinct from either type 1 or type 2 phosphatases. Limited tryptic digestion of SMP-III modulates the phosphatase activity such that it behaves like a typical type 1 phosphatase. The change in enzymatic properties correlates with the digestion of the C terminus of the catalytic subunit. SMP-III activity is modulated by a variety of reagents including divalent cations, nucleotides, NaF, and KCl. The myosin binding properties of SMP-III were determined and compared with other smooth muscle myosin phosphatases and recombinant protein phosphatase. SMP-III and SMP-IIIc both bind strongly to myosin with Kbinding of 1.9x106 M-1 and 1.9x105 M-1 respectively. These results suggest that a regulatory subunit is not required for the association of the phosphatase with myosin. The effect of various reagents on Kbinding was determined. GTP caused a 1.4 and 4 fold increase in the Kbinding of SMP-III and SMP-IIIc, respectively. The effect of GTP on the activity of SMP-III was examined. GTP inhibited SMP-III activity towards MLC20 and HMM with an IC50 of 400 µM at pH 7.4 and 150 µM at pH 7.0. The mechanism for the effect of GTP was examined. GTP may inhibit SMP-III activity by associating with the 20,000 Da myosin lightchains. A second possibility is that GTP increases the affinity of SMP-III for its substrate thereby decreasing the rate of dissociation and its movement from one substrate molecule to the next. The effect of protein kinase C (PK-C) phosphorylation on several properties of SMP-III was determined. PK-C phosphorylation has no effect on SMP-III activity, its sensitivity to heat stable inhibitor 2 or its binding of SMP-III to myosin.

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Degree

Doctor of Philosophy (Ph.D.)

Department

Biochemistry

Program

Biochemistry

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