INTRACELLULAR MITOCHONDRIAL VARIATION IN ENZYME ACTIVITY AS SHOWN BY HISTOCHEMICAL STUDIES USING LIGHT AND ELECTRON MICROSCOPY

Abstract

Activities of reduced diphosphopyridine nucleotide (DPNH)-tetrazolium reductase, succinic dehydrogenase, and reduced triphosphopyridine nucleotide-tetrazolium reductase were studied using the substrates glutamate, iso-citrate, and DPNH for the first enzyme; succinate; and 6-phosphogluconate, glucose-1-phosphate, and TPNH for the last enzyme system. These activities were quantitated in light microscopical studies by counting the formazan particles in non-vacuolated epidermal meristematic cells of timothy grass seedling roots. Using Nitro-blue tetrazolium as the H⁺-acceptor, the average counts per cell were: 57, DPNH-tetrazolium reductase; 65.5, succinic dehydrogenase; 78.2, TPNH-tetrazolium reductase. These significantly different counts were higher than those found in an earlier study using neotetrazolium, but the three relative levels of activity were unchanged. One discrepancy was that 6-phosphogluconate and succinate gave the same activity levels although 6-phosphogluconate is believed to be oxidized via TPN-linked systems. INT also was tested, but the results suggested non-specific enzyme activity and those data were discarded. Electron microscopical studies were reported in which DPNH-tetrazolium reductase activity was shown to be restricted to the mitochondrial membranes in about half the mitochondrial population of individual cells. Since preliminary counts of mitochondrial profiles from electron micrographs indicated about 1000 mitochondria per cell, the absolute numbers of formazan particles using light microscopy probably is an unreliable measure. Also, clumping of mitochondria after histochemical incubation was demonstrated in some of the electron microgaphs. This suggested that lower brightfield counts might result since one or more mitochondria may have been associated as a single formazan deposit. Generally, the data verified the earlier conclusions that (a) mitochondria were the specific site of this oxidative enzyme activity, and (b) that only a fraction of the mitochondrial population of a cell was enzymatically active for this enzyme.