Tyrosine protein kinase activity during embryogenesis.
The activity of tyrosine-specific protein kinase was estimated during early embryonic development of the sea urchin, Strongylocentrotus purpuratus, using the synthetic peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly as a probe. The peptide was not phosphorylated by the purified cyclic AMP-dependent protein kinase, phosphorylase kinase, glycogen synthase kinase 3, or casein kinase 2 but was phosphorylated by the purified epidermal growth factor-receptor kinase from A-431 cells. The sea urchin tyrosine protein kinase activity was determined in a particulate fraction (17,000 x g pellet) and in a membrane fraction obtained after discontinuous sucrose gradient centrifugation; these fractions contained higher specific activities of the kinase than the cytosolic or nuclear fractions. Unfertilized eggs had very low tyrosine protein kinase activity (0.22 pmol of peptide phosphorylated per mg of protein/min) in the particulate fraction, but the activity increased 2.5-fold by 1 h after fertilization and almost 20-fold by the gastrula stage. The enzyme activity of the membrane fraction increased similarly during this time period. The tyrosine protein kinase had an apparent Km of 8.9 mM for the peptide, and showed one-half-maximal velocity at about 35 microM MgATP. The phosphorylation of membrane fractions in vitro at different stages of embryonic development resulted in nine endogenous protein-staining bands (Mr = 26,000 to greater than 200,000; 10% Na dodecyl SO4 gels) which contained phosphotyrosine; some of the bands incorporated 32P differentially as a function of development. In the unfertilized egg membrane fraction, none of the protein-staining bands were shown to contain detectable 32P-tyrosine. Thus, fertilization results in increases in tyrosine-specific protein kinase(s) activity which continues to increase during early embryonic development; it is suggested that these protein kinases have a functional role during early development and differentiation.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D005260:Female; D005306:Fertilization; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010063:Ovum / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D012617:Sea Urchins / Q000196:embryology*; D014443:Tyrosine / Q000378:metabolism
D008565:Membrane Proteins; D009842:Oligopeptides; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases