Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma.
Protein tyrosine kinase (PTK) activities in methyl nitrosourea (MNU)-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was almost equally distributed between the particulate and soluble (cytosolic) fractions at 110,000 X g. The activity of the particulate enzyme was stimulated by non-ionic detergent Triton X-100 by about 2-fold whereas the detergent had no effect on the cytosolic form. More than 60% of the particulate enzyme could be solubilized by 5% Triton X-100. Although, both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, poly (glu: tyr; 4:1) was the best substrate (apparent Km. 0.7 mg/ml). Both forms of enzymes utilized ATP as the phosphoryl group donor, with an apparent Km of 40 microM. Among various divalent cations tested, Co2+, Mn2+ and Mg2+ were able to fulfill the divalent cation requirement of both forms of the PTKs. All these cations exerted biphasic effects on the kinase activities, however, Mg2+ was the most potent cation. Agents such as epidermal growth factor, insulin and platelet derived growth factor which stimulate their respective receptor-PTK activities were without effect on PTK activities of mammary carcinoma. On the other hand, though heparin and quercetin inhibited both enzyme activities in a concentration dependent manner, the particulate form was more sensitive to inhibition than the cytosolic form. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms of PTKs. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play an important role in mammary carcinogenesis.
Research Group on Diabetes and Metabolic Regulation, University of Montreal, Québec, Canada.
A K
AK
Srivastava
J C
JC
Chiasson
J L
JL
Chiasson
A
A
Lacroix
L
L
Windisch
D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D003902:Detergents; D005260:Female; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000139:chemically induced / Q000188:drug therapy / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008770:Methylnitrosourea; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D003902:Detergents; D008670:Metals; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D008770:Methylnitrosourea; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases