Abstract
Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.
Highlights
From the DeDartment of Pharmacobm and Moss Heart Center, University of Texas Health Science Center at Dallus, -I
The molecular weights of the purified striated muscle myosin light chain kinases (M, = 75,000-95,000) were significantly lower than thepurified smooth muscle kinases (M, = 130,000-155,000). It has been suggested (Walsh and Guilleux, cardiac, andchicken gizzard smooth muscle myosinP- 1981; Guerriero et al, 1981) that the size heterogeneity of light chains were very similar for the chicken and myosin light chain kinase was due to proteolysis during the rabbit skeletal muscle myosin light chain kinases
There was no evidence of proteolysis of againstchickengizzard myosin lightchain kinase. the antibody-labeled myosin light chain kinases, and none of Proteolytic digestion followed by sodium dodecyl sul- the labeled polypeptides were as large as the purified marnfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides
Summary
From the DeDartment of Pharmacobm and Moss Heart Center, University of Texas Health Science Center at Dallus, -I. The slurry was stirred for 10 min and collected on a Anti-myosin Light Chain Kinase Antiserum Preparation and Charsintered glass funnel and washed with 400-500mlof the elution acterization-Antiserum to rabbit skeletal muscle myosinlight chain buffer. EDTA, 0.5 mM dithiothreitol and 3 mM NaN3.The column fractions Chicken or rabbit skeletal muscle myosin light chain kinase (0.2 nM (4 ml each) were assayed for kinase activity, and thepeak of activity final concentration) was incubated for 1h a t 30 "Cin the presence of was pooled. One-dimensionalPeptide Mapping-Chicken skeletal, rabbit skeletal, and chicken gizzard smooth muscle myosin light chain kinases (-2 pglwell) were subjected to SDS-polyacrylamide electrophoresis on 7.5% polyacrylamide slab gels according to Laemmli (1970).The gelswere briefly stained (10 min)in 0.2%Coomassie Blue, 50% methanol, 10%acetic acid. The bands were excised and placed in microfuge tubes containing1ml of 0.125 M Tris-HC1,pH 6.8,0.1% SDS and frozen at -20 'C
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