A denaturant-insoluble form of tyrosine hydroxylase in PC12 pheochromocytoma cells.
A 6M urea-insoluble form of tyrosine hydroxylase (THi) was detected in PC12 pheochromocytoma cells by western blotting immunodetection methods, and the characteristics and mechanisms of formation of this insoluble species were investigated. THi accounts for about 4% of the immunodetectable tyrosine hydroxylase in exponentially dividing pheochromocytoma cells. It is unlikely that a subpopulation of dead or dying cells is the source of THi since essentially no changes in THi levels were detected when cell death was intentionally increased. To measure the kinetics of formation of cellular THi, exponentially dividing cells were metabolically labeled first with [3H]leucine and then with [14C]leucine, and though both 3H and 14C were incorporated into soluble tyrosine hydroxylase, the near absence of 14C in THi demonstrated that a lag period of at least a day exists between biosynthesis of tyrosine hydroxylase and the accumulation of measurable THi. The cellular accumulation of THi can evidently be regulated by the cell, since upon nerve growth factor (NGF) treatment of cells the total content of tyrosine hydroxylase increased and the content of THi decreased to yield, overall, a fivefold lower proportion of THi after 4 days. A large increase in urea-insoluble enzyme was found upon sublethal exposure of cells to ferrous ion and hydrogen peroxide, indicating that oxidative damage via metal-ion-catalyzed formation of hydroxide free radical can yield an enzyme that is similar in its insolubility to THi.
Fleming-Jones
R M
RM
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.
McFadden
P N
PN
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D009414:Nerve Growth Factors / Q000494:pharmacology; D010084:Oxidation-Reduction; D016716:PC12 Cells / Q000201:enzymology*; D011489:Protein Denaturation; D051381:Rats; D012995:Solubility; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D009414:Nerve Growth Factors; D014508:Urea; D014446:Tyrosine 3-Monooxygenase