Pitfalls in the light microscopical detection of NADH oxidase

Abstract

NADH oxidase activity has been detected at the ultrastructural level using cerium ions to trap H2O2 generated by the enzyme (via intermediate reactive oxygen species). In an attempt to localize NADH oxidase activity at the light microscope level using the cerium-diaminobenzidine (DAB)-nickel-H2O2, the cerium-DAB-cobalt-H2O2 or the cerium-alkaline lead procedures, the distribution patterns of the revealed enzyme were found to be identical to those for non-specific alkaline phosphatase and especially 5'-nucleotidase activity. With the cerium-DAB-cobalt-H2O2 visualization procedure, the distribution pattern of the final reaction product was similar to that obtained with the other two techniques but much less final reaction product was formed. Incubations for NADH oxidase activity performed in the presence of exogenous catalase or in the absence of catalase or peroxidase inhibitors did not affect the staining intensity, whereas inhibitors of 5'-nucleotidase (EDTA) and non-specific alkaline phosphatase (levamisole) always did. Therefore, phosphatases contribute to the formation of the final reaction product. Since NADH initially cannot be hydrolysed by either of these two phosphatases, then presumably nucleotide pyrophosphatase (E.C.3.6.1.9) cleaves NADH into 5'-AMP and nicotinamide mononucleotide in a first step. Both nucleotides can be hydrolysed further by the two monophosphatases. These then generate cerium phosphate which is detected by the DAB-nickel-H2O2, DAB-cobalt-H2O2 or lead visualization methods.

Cerium methods for light and electron microscopical histochemistry

“…In contrast, in the presence of levamisole, an inhibitor of alkaline phosphatase, and/or EDTA, an inhibitor of 5'-nucleotidase, formation of final reaction product was decreased. Apparently, these phosphatases with a rather broad substrate specificity can generate phosphates as follows: membranebound nucleotide pyrophosphatase hydrolyses NADH (Parson, 198 7) with concomitant 5'-AMP production which in turn can be hydrolysed by alkaline phosphatase and 5'-nucleotidase (Gossrau et al, 1990b). On the other hand, when demonstrating phosphatase activity with cerium, there is the remote possibility that formation of hydrogen peroxide may produce a false positive reaction.…”

A new staining method for the detection of activities of H2O2‐producing oxidases on gels and blots using cerium and 3,3′‐diaminobenzidine

“…Recently however, Angermuller and Fahimi [12] showed that this pale reaction product can be converted to a dark-brown precipitate by the addition of 3,3'diaminobenzidine (DAB), which is oxidized by cerium perhydroxide to its polymeric form. This approach has found broad application in light microscopic localization of oxida-ses in tissue sections [13,14].To ourknowledge this method has neither been used for the identification and biochemical characterization of oxidases on native gels after electrophoretic separation nor on corresponding nitrocellulose blots.…”