title: Properties and mechanism of action of viral and cellular tyrosyl protein kinases.
abstract: We have demonstrated the usefulness of several angiotensin analogs as substrates for a number of tyrosyl protein kinases of viral or cellular origin. Results of initial rate studies with pp60src at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 microM respectively and Vmax was 1.0 nmol/min/mg. The end-product ADP and the ATP analog, AMP-PNP, were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]-angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. The available evidence allowed us to conclude that while pp60src contained essential histidine and lysine residues in its active site, the kinase reaction does not involve a phosphoryl enzyme intermediate. Phosphorylation of the angiotensin peptides in vitro also has allowed us to demonstrate the presence of at least two tyrosyl protein kinases in the cytoplasm of normal rat liver cells. These kinases appear to be novel in that they are present in normal cells and are not stimulated by growth factors. Also, results of preliminary experiments indicate that these kinases are not immunologically related to the transforming gene products of Rous and Fujinami sarcoma viruses (unpublished observations). The identification of these new kinases represents another application of the angiotensin peptides as substrates for tyrosyl kinases (13). The results obtained do not exclude the possibility that there exist in rat liver other tyrosyl kinases that do not phosphorylate these particular peptide substrates. No attempt has been made to characterize tyrosyl kinases associated with the plasma membrane fraction. Although they represent only a small fraction of the total activity of liver cells, the plasma membrane kinases have a relatively high specific activity. These kinases may be identical with growth factor receptor kinases previously identified in liver cell membranes (5). The most abundant tyrosyl kinase in rat liver cytoplasm has a molecular weight of 75,000 daltons and was found in cytosol and microsomal salt-wash fraction. The observation that the purified 75 Kd enzyme phosphorylates a 75 Kd protein on tyrosine residues suggests that the enzyme may possess autophosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000201:enzymology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D015688:Oncogene Protein pp60(v-src); D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D014764:Viral Proteins; D000804:Angiotensin II; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)