IMPROVED METHOD FOR HISTOCHEMICAL DEMONSTRATION OF PHOSPHOGLUCOMUTASE

Abstract

A method for the histochemical demonstration of phosphoglucomutase was improved in cryostat sections of liver and muscle of rabbit under light microscope.Several biochemical activators and a specific inhibitor for the enzyme were applied. Magnesium ions (1 mM) and L-histidine (5 mM) had an advantage of histochemical demonstration of the enzyme activity. The specific inhibitor, beryllium ions (1 mM) were effective to prove histochemically the specificity of the enzyme. Imidazole buffer (4×10-2M pH 7.4) was qualified for the incubation of demonstrating the enzyme reaction.Suitable conditions for histochemical reaction for the enzyme activity were determined as follows: 80 mg glucose-1-phosphate, 5 mg adenosine triphosphate, 2.5 mg nicotinamide adenine dinucleotide phosphate (NADP), 1 mM magnesium chloride, 5 mM L-histidine, 5 ml nitro blue tetrazolium (Nitro BT, 1 mg/ml), 10 ml imidazole buffer (pH 7.4), 5 ml 3% gelatin, 0.04 ml glucose-6-phosphate dehydrogenase solution (1 mg/ml) and distilled water in a total volume of 25 ml. Two control procedures were prepared, using the incubation mixture without glucose-1-phosphate and the substrate mixture with 1 mMberyllium sulphate.The reaction products, dark blue granules of diformazan were intensely demonstrated in cytoplasms of rabbit liver cells in all areas of lobuli, without nuclei. They were moderately revealed in intermyofibrillar sarcoplasms of the large fibers and weakly in the small fibers.