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QUANTITATIVE CHARACTERIZATION OF A HISTOCHEMICAL ENZYME SYSTEM
1 Section on Histochemistry, Laboratory of Experimental Pathology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland
The renal alkaline 
-naphthyl phosphatase has been studied in four assay systems ranging from the biochemical assay (homogenates incubated with substrate in the absence of diazonium salt) to the histochemical assay (frozen sections incubated with substrate in the presence of the diazonium salt, fast violet B). Quantitative and kinetic analyses were carried out for the purpose of determining how several factors influence the histochemical assay.
It was found that the stabilized diazonium salts partially inhibit as well as alter the kinetic characteristics of the phosphatase reaction. The steady state is rapidly reached in the histochemical system so that no lag period is observed, indicating that the coupling reaction and the penetration of substrate into the section are rapid processes. However, the evidence indicates that, especially at low substrate concentrations, a steady state substrate concentration gradient in the section is established so that the concentration of substrate in the section is less than it is in the medium. Thus, high substrate concentrations are required to demonstrate the total enzymic activity in a tissue section. Studies of this nature would appear to be prerequisites to any attempts at photometric quantitation of enzymic histochemical reactivities in tissue sections or zymograms.
Submitted on August 26, 1963
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