KINETICS OF ENZYME ACTION AND INHIBITION IN INTACT TISSUES AND TISSUE-SLICES, WITH SPECIAL REFERENCE TO CHOLINESTERASE

If substrate diffusion is a rate-limiting step in enzymic reactions in intact tissues or tissue slices, a substrate gradient will develop within the tissue when the enzyme is assayed by immersion of the intact tissue in a solution of substrate. The existence of such substrate gradients does not alter the apparent Vmax, but does cause deviation from normal Michaelis-Menten kinetics, and raises the apparent Km. Provided no complications are introduced due to substrate inhibition, the activity of the intact tissue at sufficiently high substrate concentration will equal that of the homogenate unless some active sites are inaccessible to the substrate in the intact tissue due to permeability barriers. At low substrate concentration, the activity of the intact tissue will always be less than that of the homogenate and the ratio of the 2 will depend on the kinetic parameters (Vmax and Km) of the enzyme in the homogenate, the diffusion coefficient of the substrate in the tissue, and the shape and size of the intact tissue. The altered kinetic behavior of enzymes in intact tissues may also have a profound effect on the apparent degree of inhibition produced by enzyme inhibitors. The substrate gradients which may develop when a tissue is bathed with an exogenous substrate which is a neurotransmitter would not exist when that transmitter is liberated locally in synaptic regions by nerve stimulation. This may provide a basis for understanding the differences which are sometimes found between the effect of drugs on responses to nerve stimulation and on responses to exogenously applied neurotransmitter.