LETTERS TO THE EDITOR ON THE SPECIFICITY OF HISTOCHEMICAL DEMONSTRATION FOR GLUCOSEPHOSPHATE ISOMERASE D-Glucose-6-phosphate ketol-isomerase, i.e. glucosephosphate isomerase has an important function in the regulation of carbohydrate metabolism , representing a branching point between the glycolytic pathway and the pentosephosphate path- way. Since this enzyme was discovered in 1933 by Lohmann (Biochem . Z. 262: 137.) in extracts prepared from yeast and animal tissues, it's biological property had been investigated by Noltman et at. (Biochem Z. 331: 436, 1959), Baich et at. (J. Biol. Chem. 235: 3130, 1960), and others (Topper. J. Biol. Chem. 225: 419, 1957; Tsuboi et at. J. Biol. Chem. 231: 19, 1958; Kahana et at. J. Biol. Chem. 235 : 2178, 1960). On the other hand, a new method for histochemical demonstration of this enzyme was reported by Yano (J. Kumamoto Med. Soc. 43 : 729, 1969), using histo- chemical analytical procedures. The same method in which the histochemical system is similar was separately reported by Meijer and Bloem (Acta histochem. 32: 110, 1969) . Using a gel film method for coupled enzyme reaction, Sigel and Pette reported another method (J. Histochem. Cytochem. 17 : 225, 1969). Recently, Orchardson and McGadey (Histochemie, 22: 136, 1970) also reported a method for this enzyme. A histochemical method has in general been established to demon- strate one enzyme activity by re-examination and it's specific distribution in tissue cells. Accordingly, it must be tried here to confirm the specificity of histochemical reaction of glucosephosphate isomerase. As reported by Yano (J. Kumamoto Med. Soc. 43: 729, 1969; Acta histochem. Cytochem. 2: 147, 1969: Kumamoto Med. J. 23:103, 1970), many problems were investigated such as follows : (1) many conditions in specific reaction, using histochemical analytical procedures, (2) effect of 6-phosJ phogluconate as a competitive inhibitor on this enzyme activity, (3) histochemical comparison with this enzyme and the related enzymes in rabbit skeletal muscles, and (4) carbohydrate metabolism in ascites hepatomas AH 13 cells and AH 39 cells. As shown in Table 1, the results obtained revealed that the histochemical reaction appeared to be strikingly positive in condition (I), moderately positive in condition (V), and negative in conditions (II), (III) and (IV). The reaction in condition (VI) appeared relatively positive. It was thought under the condition (V) that the positive reaction may result from a presence of endogenous glucose-6- phosphate dehydrogenase in tissue cells, but the substrate in the reaction of endogenous glucose-6-phosphate dehydrogenase may be converted from fructose-6- phosphate by the catalysis of glucosephosphate isomerase, contrary to conditoin (IV). Meijer and Bloem also reported on the specificity by incubating sections in a medium with-out the addition of exogenous glucose-6-phosphate dehydro- genase that the activity was found only in sections containing this enzyme endogenously. Since no other enzyme was known to convert fructose-6-phosphate into glucos-6-phosphate, the formation of glucose-6-phosphate must depend exclusively on the activity of glucosephosphate isomerase. 103 104 TABLE 1 Control procedures of specific reaction for glucosephosphate isomerase S. strikingly M. moderately W. weakly P. positive N. negative 6-Phosphogluconate was examined as an only utilizable inhibitor with equimolecular concentration (10 mM) to the substrate, and the activity of this enzyme was slightly inhibited, as shown in condition (VI). 1 mM and 5 mM of 6-phos- phogluconate were not revealed to inhibit this enzyme reaction. Complete inhibi- tion with 6-phosphogluconate would not occur histochemically, because of in- complete inhibitory action, as Parr reported previously (Nature 178: 1401, 1956; Biochem. J. 65: 34, 1957). Although Meijer and Bloem investigated the inhibitory effect of inhibitors (deoxyglucose-6-phosphate, sorbitol-6-phosphate), upon the enzyme activity, reliable data on a possible inhibitory effect on the activity of the enzyme could not be obtained with them. This inhibition was found to be de- pendent not only on the concentration of the inhibitor but also on the concentration of the substrate. With relation to this enzyme, NADPH dehydrogenase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase were histochemically investigated in rabbit skeletal muscles (Yano. J. Kumamoto Med. Soc. 43: 729, 1969). The activity of this enzyme was, in serial sections, demonstrated in muscle fibers in which NADPH dehydrogenase activity was demonstrated, as well as phos- phoglucomutase. It was found from this fact that this enzyme was very dependent upon NADPH dehydrogenase activity. The enzyme was shown, by Orchardson and McGadey on the distribution of this enzyme, to be widely distributed, con- firming biochemical findings. On the specific distribution of this enzyme in tissue cells, the author compared this enzyme and the related enzymes in rabbit muscles (J. Kumamoto Med. Soc. 43: 729, 1969) and studied the carbohydrate metabolism in Yoshida ascites hepatoma AH 13 cells and AH 39 cells (Acta histochem. cytochem. 2 : 147, 1969). It was found from these results that the enzyme was very dependent upon NADPH dehydrogenase, and that in the carbohydrate metabolism of tumor cells, this enzyme showed specifically the activity which represented the characteristic property of both tumor cells. Although it is well known that AH 13 is a glycogen-rich strain and AH 39 105 is a glycogen-poor strain, glycolytic activity shown by glucosephosphate isomerase, aldolase and lactate dehydrogenase was higher in AH 39 cells than in AH 13 cells. This fact was closely related to the findings in which the glycogen granules were deposited more abundantly in AH 13 cells than in AH 39 cells. On the other hand, Meijer and Bloem reported from a biopsy of musculus quadriceps obtained from a patient with myopathy that the muscle tissue showed occasional fibers in which the activity of this enzyme could not be demonstrated locally, while the activity of mitochondrial enzyme was found in these areas. How- ever, it was considered in doubt from my own investigation that only one muscle fiber in myopathy was selectively affected, showing inactivity of glucosephosphate isomerase. As shown in Table 2, an attempt was made to compare with the components of the substrate mixtures after Mejier and Bloem, Yano and Orchardson & McGadey. The substrate mixture which Meijer and Bloem reported was similar to Yano's own mixture apart from control procedures, while Orchardson and McGadey added MgSO4 to the substrate mixture, without the addition of exogenous glucose-6- phosphate dehydrogenase. Although the addition of MgSO4 to the substrate mixture was considered to activate the enzymatic reaction of glucosephosphate isomerase, the histochemical reaction only appeared to be moderately positive in condition (V), as mentioned above. Glucosephosphate isomerase was more intensely detected in AH 39 cells and AH 13 cells than phosphoglucomutase which is demonstrated by the same procedure with the same mechanism for demonstrating enzyme. In spite of the histochemical difference between both enzymes, glucose- 6-phosphate dehydrogenase activity in both strains was similarly demonstrated itself. Therefore, the addition of exogenous glucose-6-phosphate dehydrogenase to the substrate mixture was concluded to be required to prevent the activity of glucose- TABLE 2 Components of substrate mixture * : Control procedures are important . 106 phosphate isomerase from the restriction of endogenous glucose-6-phosphate dehydrogenase activity. In addition, phenazine methosulfate (PMS) with potassium cyanide was added to the incubating mixture, because it would promote an increase in the formazan deposition and demonstrate this enzyme activity independently of NADPH dehydro- genase activity in tissue cells. According to this procedure, this enzyme activity was demonstrated strikingly in both large and small fibers of rabbit skeletal muscles. Differences in enzyme activities among muscle fibers should be considered to have a physiological significance, although Sigel and Pette repeated this fact. It was distinctly recognized in conclusion that the histochemical techniques for glucosephosphate isomerase may be usefull and specific, although many designated conditions should be tried to confirm the specificity of this enzyme reaction, because the histochemical system of demonstrating this enzyme is intricate with requirement of catalysis of two intermediate enzymes (glucose-6-phosphate dehydrogenase and NADPH dehydrogenase). February 24, 1971 Yoshinobu Yano, M. D. Laboratory of Clinicopathology, Kumamoto National Hospital Kumamoto.