Inhibition of proteinase K activity by copper(II) ions.
Disease-related prion protein, PrPSc, can be distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Several studies have suggested that copper(II) ions can convert PrPC to a proteinase K-resistant conformation; however, interpretation of these studies is complicated by potential inhibition of proteinase K (PK) by copper(II) ions. Here we have examined directly the kinetic and equilibrium effects of copper(II) ions on PK activity using a simple synthetic substrate, p-nitrophenyl acetate. We show that at equilibrium two to three copper(II) ions bind stoichiometrically to PK and destroy its activity (Kd < 1 microM). This inhibition has two components, an initial reversible and weak binding phase and a slower, irreversible abolition of activity with a half-time of 6 min at saturating copper(II) ion concentrations. Copper(II) ions produce a similar biphasic inhibition of PK activity in the presence of brain homogenate but only when the copper(II) ion concentration exceeds that of the chelating components present in brain tissue. Under these conditions, the apparent resistance of PrPC to proteolysis by PK appears to be directly attributable to the inhibition of PK activity by copper(II) ions.
Stone
Lisa A
LA
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, U.K.
Jackson
Graham S
GS
Collinge
John
J
Wadsworth
Jonathan D F
JD
Clarke
Anthony R
AR
D000818:Animals; D001921:Brain / Q000378:metabolism; D003300:Copper / Q000494:pharmacology*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018621:PrPC Proteins / Q000378:metabolism; D018620:PrPSc Proteins / Q000378:metabolism; D013997:Time Factors
D018621:PrPC Proteins; D018620:PrPSc Proteins; D003300:Copper; D019286:Endopeptidase K