Michaelis constants for human brain monoamine oxidase have been determined with tyramine, benzylamine and dopamine as the substrates. In each case double reciprocal plots were linear over a 20-fold range of substrate concentrations. The method of mixed substrates failed to indicate heterogeneity in the enzyme preparation. The theory of the method of mixed substrates has been extended to cover systems in which two enzymes are each active toward two different substrates. It is shown that, regardless of the difference in Km values of each of the individual enzymes for the two substrates, if the Km values of the two enzymes are similar for each individual substrate the situation is indistinguishable from the case in which only a single enzyme is present. The observation that the mixed substrate experiments do not indicate the presence of more than one species of monoamine oxidase cannot therefore be regarded as providing firm evidence for homogeneity.
