Cytochemical localization of two glycolytic dehydrogenases in white skeletal muscle.
The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.
D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006651:Histocytochemistry; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008854:Microscopy, Electron; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D009243:NAD; D010088:Oxidoreductases / Q000378:metabolism; D010619:Phenazines; D011817:Rabbits; D013778:Tetrazolium Salts
D010619:Phenazines; D013778:Tetrazolium Salts; D009243:NAD; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases