Isolation and characterization of tropomyosin kinase from chicken embryo

• Published in: Biochimica et biophysica acta Vol. 925; no. 1; pp. 17 - 26
• Main Authors: deBelle, Ian, Mak, Alan S.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16.07.1987; Elsevier; North-Holland
• Subjects: Adenosine Triphosphate; Adenosine Triphosphate - metabolism; Age Factors; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chick Embryo; Chick Embryo - enzymology; Chicken embryo; chicks; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; embryo (animal); Enzymes and enzyme inhibitors; enzymology; Fundamental and applied biological sciences. Psychology; isolation & purification; kinases; Kinetics; metabolism; Molecular Weight; Muscle Proteins; Muscle Proteins - isolation & purification; Muscle Proteins - metabolism; Muscles; Muscles - enzymology; phosphotransferases (kinases); Protein Kinases; Protein Kinases - isolation & purification; Protein Kinases - metabolism; purification; Rabbits; Rabbits - metabolism; Species Specificity; Substrate Specificity; Transferases; Tropomyosin; Tropomyosin - metabolism; Tropomyosin kinase; Tropomyosin kinase; SDS; PMSF; Pipes; Chicken embryo; EGTA; DFP; Vertebrata; Embryo; Chicken; Aves; Enzyme; Catalyst activity
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/0304-4165(87)90143-7

Tropomyosin kinase is partially purified from 4-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of M r = 76 000 and 43 000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250 000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent K m for skeletal α-tropomyosin and ATP is 50 μM and 200 μM, respectively V max varies between 100–300 nmol/min per mg depending on the purity of the preparation. Mg 2+ and dithiothreitol are essential for activity but Ca +, calmodulin and cAMP are not required. The optimum temperature is 37°C and optimum pH is about 7.5 Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.