W. Taylor, P. Andrews, C. K. Henrikson
Jun 1, 1980
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Analytical biochemistry
Abstract
Abstract Several compounds having the general formula, Z· X - l -prolyl-4-methoxy-β-naphthylamide have been examined as substrates for a homogeneous rat brain proline endopeptidase. The compounds examined included X equal to l -Ala-, l -His, l -Lys-, and l -Asp-, the latter compound being the poorest substrate of those examined. A simple assay was developed based upon the appearance of the fluorescent product, 4-methoxy-β-naphthylamine. The assay is highly sensitive, continuous, and affords the possibility of utilizing the product of the reaction for tissue or subcellular localization of the enzyme. The homogeneous enzyme hydrolyzed each substrate examined. The most favorable kinetic constants were noted with Z- l -Ala-, Z- l -His-, and Z- l -Lys, Pro-4-methoxy-β-naphthylamide. These substrates show a pH optimum at 8.3 with the homogeneous rat brain proline endopeptidase. The tissue distribution of the enzyme was examined using each substrate and the highest specific activity was noted in the brain. A monospecific antibody prepared against the homogeneous rat brain enzyme inactivates essentially all enzyme activity in every tissue examined except kidney, where as musch as 7% residual activity was noted.