OBSERVATIONS ON THE SUBCELLULAR LOCALIZATION OF OXIDATIVE ENZYMES WITH NITRO BLUE TETRAZOLIUM

Abstract

The effectiveness of nitro blue tetrazolium in localizing enzymes at the subcellular level has been investigated. The primary source of interference with enzyme localization was lipid material which progressively accumulated formazan deposits during incubation in a tetrazole medium containing pyridine nucleotide. Acetone fixation at 2°C extracted this lipid and permitted the visualization of both mitochondrial and microsomal enzymes. There was no evidence for an unspecific adsorption of Nitro-BT on lipoprotein membranes which led to an antifactual staining pattern. The demonstration of oxidative enzyme activity in the alkaline pH range was not impeded by "nothing dehydrogenase" if fats had been removed. Otherwise, lipid-tetrazole interactions contributed heavily to nonenzymatic formazan deposition which was difficult to distinguish visually from mitochondrial activity. MTT-Co++ was also adsorbed by lipids. After cold acetone fixation, patterns of this formazan were not equivalent to those found with Nitro-BT. The relation of these findings to the current evaluations of enzyme activity at the subcellular level is discussed. At the level of the light microscope, nitro blue tetrazolium is an adequate indicator of the intracellular sites of oxidative enzyme activity if lipid interference is counteracted.