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QUANTITATION OF TISSUE-BOUND RENAL AMINOPEPTIDASE BY A MICRODENSITOMETRIC TECHNIQUE

K. FELGENHAUER 1 and G. G. GLENNER 1

1 Laboratory of Experimental Pathology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland

Relationships between biochemical and histochemical assay systems can be evaluated by appropriate techniques. With rat kidney as the test object the enzyme localized in the tissue section hydrolyzing l-leucyl-beta-naphthylamide (LNA) was found to be identical with a purified particle-bound, cobalt-activated aminopolypeptidase previously characterized biochemically. A soluble sulfhydryl-dependent aminopeptidase, present in the rat kidney and also known to hydrolyze LNA, was not demonstrated in the histochemical system, although the diazonium salt employed incidentally caused the insolubilization in the tissue section of a significant proportion of previously extractable, particle-bound enzyme. Linearity between the enzymic activity present and the measurable enzymic activity was demonstrated to exist at the substrate concentration, pH level, section thickness and diazonium salt concentrations used in the assay procedure, but did not exist when these factors were varied beyond certain well defined limits. In addition, the effect of diazonium salt on both the inhibition and insolubilization of enzyme in the tissue section could be quantitated. Based on this evaluation of the enzymes hydrolyzing LNA in the rat kidney, a microdensitometric assay method employing a constant flow incubating chamber was developed to characterize and quantitate the LNA-hydrolyzing enzyme in the proximal tubuli. This study defines many of the parameters necessary for the future investigation by quantitative and qualitative methods of histochemical systems capable of providing resolutions of a higher order of magnitude and the retention of a greater proportion of extractable enzyme within the tissue section.

Submitted on July 12, 1965


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