Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases.
In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, beta-glucuronidase, non specific arylesterase, microsomal arylsulphatase, beta-galactosidase, beta-N-acetylglucosaminidase, acid alpha-glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003979:Diazonium Compounds / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005404:Fixatives; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D009284:Naphthols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling / Q000379:methods
D003979:Diazonium Compounds; D004791:Enzyme Inhibitors; D005404:Fixatives; D009284:Naphthols; D006867:Hydrolases