Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis.
Catalytic properties (KM, Vmax) of aminopeptidase in pig kidney sections, in isolated membranes and in a solubilized purified form were investigated using amino acid 2-naphthylamides and 4-methoxy-2-naphthylamides. In the first case these properties were estimated on the basis of the stain intensity resulting from the coupling of product with Fast Blue B, in the latter two cases they were measured fluorometrically. The following observations were made: (1) In all three cases the substrate turnover was shown to be a direct function of time and enzyme concentration. (2) The values obtained for the solubilized and the membrane bound form were practically identical but differed from those found in tissue sections. (3) Each amino acid derivative had defined constants, but these were difficult to obtain in sections, especially if it was necessary, on account of poor solubilities, to use low substrate concentrations. (4) Hydrophilic amino acid derivatives were adsorbed to tissue membranes much less than hydrophobic ones. (5) Fast Blue B caused a non-competitive inhibition of enzymic activity. (6) Binding of antibody against pure aminopeptidase caused inhibition of the enzymic hydrolysis of all the naphthylamides. Thus, histochemical stain intensities per time and area derived from one substrate at a defined concentration are suitable for the determination of enzyme concentrations. However, no conclusions regarding the homogeneity of the enzyme in sections can be drawn by comparing the stain intensities obtained with different substrates in contrast to data from the inhibition of substrate hydrolysis by antibody.
D000577:Amides; D000626:Aminopeptidases / Q000032:analysis*; D000818:Animals; D003979:Diazonium Compounds; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D009281:Naphthalenes; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D000577:Amides; D003979:Diazonium Compounds; D009281:Naphthalenes; D000626:Aminopeptidases