ULTRACYTOCHEMICAL DEMONSTRATION OF NADPH-FERRICYANIDE REDUCTASE ACTIVITY

Abstract

As an extension of our studies on the copper ferrocyanide method for the ultracytochemical demonstration of oxidoreductase activity, the method for the detection of NADPH-ferricyanide reductase activity was developed. The incubation medium consists of 20mg of NADPH, 13.0ml of 0.1M phosphate buffer, pH 7.0, 1.3ml of 0.1M sodium citrate, 2.0ml of 30mM copper sulfate, 1.7ml of double distilled water, 2.0ml of 5mM potassium ferricyanide and 3.0g of sucrose. It was observed in the preliminary experiment that potassium sodium tartrate caused more diffusion of the final reaction products than sodium citrate.Fresh tissues from brain, liver, heart and kidney of normal adult rats were used. Formaldelyde inhibited the enzymatic activity completely. Incubation with medium was carried out for 30 minutes at 37°C under aerobic and anaerobic conditions. After the incubation specimens were fixed in 1% osmium tetroxide and processed for electron microscopy.The NADPH-ferricyanide reductase activity was extremely low in all tissues examined. In the enzymatically positive tissues the activity was observed solely in mitochondria. No activity was demonstrated in any element of the endoplasmic reticulum. The enzymatic activity in specimens incubated under anaerobic conditions was slightly higher in terms of the number of enzymatically positive mitochondria and the rate of positive reaction in each mitochondrion than that incubated under aerobic conditions.In mitochondria the reaction product was observed in the membrane system including the outer membrane, the intracristal space and the outer space. In some areas, the activity in the outer membrane was quite evident. No activity was observed in the mitochondrial matrix.