Kinetics of heat inactivation of phenyl valerate hydrolases from hen and rat brain.
Heat inactivation was studied at 45, 50, 55, and 60 degrees for all of the phenyl valerate hydrolases (PVase), including neurotoxic esterase (NTE) and inhibitor-resistant esterase (IRE), in homogenates of hen or rat brain or in preparations of hen brain microsomal membranes. Hen and rat brain homogenates were prepared in buffer (50 mM Tris/0.20 mM EDTA, pH 8.00, at 25 degrees). Hen brain microsomes were suspended either in buffer or in aqueous dimethyl sulfoxide (DMSO, 40%, w/v), or solubilized either in aqueous Triton X-100 (0.10%, w/v) or in 40% (w/v) DMSO. Enzyme activities were measured at 37 degrees using phenyl valerate as substrate. Each enzyme activity in all of the preparations exhibited biphasic heat inactivation kinetics. Apparent rate constants were calculated for the fast (kf) and slow (ks) reactions, along with the relative amounts of activity in each component (Af, As) expressed as percentages of the total activity. For a given preparation and temperature, respective values of kf or ks were similar for PVase, NTE, and IRE, with a mean kf/ks ratio of 52 across all preparations. Af and As were a function of temperature. Mean values of the apparent activation energies (Ea) for all activities and preparations were 44 and 25 kcal/mol for the fast and slow inactivation reactions respectively. These results indicate that all phenyl valerate hydrolases in hen and rat brain undergo a common heat-induced structural change leading to loss of enzymic activity.
Reiner
E
E
Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109.
Davis
C S
CS
Schwab
B W
BW
Schopfer
L M
LM
Richardson
R J
RJ
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D002645:Chickens; D004789:Enzyme Activation; D005260:Female; D006358:Hot Temperature; D007700:Kinetics; D008297:Male; D051381:Rats
D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase