STUDIES OF CYTOCHEMICAL LOCALIZATION OF SPECIFIC DEHYDROGENASES IN FROZEN,DISEASED TISSUES OF PINUS

Intracellular activity of individual dehydrogenases in frozen tissues of Pinus monticola and Cronartium ribicola was demonstrated by supplying a specific substrate and the appropriate pyridine-nucleotide-linked coenzyme. Freezing broke cell permeability barriers releasing endogenous coenzymes and substrates which had produced nonspecific enzymatic reduction of nitro blue tetrazolium by miscellaneous dehydrogenases throughout fresh tissues. Freezing enhanced specificity by accentuating the differences between control and treatment sections. Succinic, ethanol, glutamic, α-glycerophosphate, isocitric, lactic, malic, glucose-6-phosphate, and 6-phos-phogluconate dehydrogenases and NAD and NADP diaphorases were localized within cells of the blister rust fungus and its western white pine host. NAD- and NADP-linked forms of glutamic, isocitric, and malic dehydrogenases were also detected. The distribution and activity of the enzymes are described for cell types of host and pathogen. β-Hydroxybutyric and pyruvic dehydrogenases were not detected. Calcium and magnesium (5 × 10^?3 m final conen) and zinc (1.5 × 10^?5 m final concn) had little or no effect on localization. Amytal increased reduction by 6-phosphogluconate, glutamic, and ethanol dehydrogenases while azide depressed the reaction for the last enzyme. Cyanide augmented diformazan formation with succinate. Transhydrogenase was eliminated as a likely contributor to spurious localization in these frozen tissues. Enzymatically produced diformazan appeared on the surface of lipid droplets in cells of both organisms in fresh and frozen sections. The use and interpretation of data from frozen and fresh tissues in tetrazolium cytochemistry are discussed.