Summary
The reaction velocity of glucose-6-phosphate dehydrogenase has been quantified by continuous monitoring on a Vickers microdensitometer of the reaction product as it formed in sections of different thickness of rat tracheal epithelium. Reaction velocity was directly proportional to section thickness when either tetranitro BT or neotetrazolium was used as the final acceptor; the rate was the same with each tetrazolium salt.
However, the amount of formazan deposited in a given time was not proportional to section thickness. When tetranitro BT was employed the reaction became non-linear in the thicker sections due to the inability of the instrument to record beyond a certain absorbance value. Using neotetrazolium a lag phase, due to the failure to overcome the critical supersaturation level of the formazan, preceded the linear response. The duration of this phase decreased as section thickness increased.
The implications of these findings on studies using conventional “end point” methods of measurement are discussed.
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References
Altman FP (1976) Tetrazolium salts and formazans. Prog Histochem Cytochem 9(3):1–56
Butcher RG (1972) Precise cytochemical measurement of neotetrazolium formazan by scanning and integrating microdensitometry. Histochemistry 32:171–190
Butcher RG (1978) Oxygen and the production of formazan from neotetrazolium chloride. Histochemistry 56:329–340
Butcher RG, Altman FP (1973) Studies on the reduction of tetrazolium salts. II. The measurement of the half-reduced and fully reduced formazans of neotetrazolium chloride in tissue sections. Histochemistry 37:351–363
Butcher RG, Van Noorden CJF (1984) Reaction rate studies of glucose-6-phosphate dehydrogenase activity using four different tetrazolium salts. J Histochem Cytochem (in press)
Evans AW (1979) Quantitative cytochemistry of glucose-6-phosphate, lactate and succinate dehydrogenases in oral mucosa during hamster cheek pouch cardinogenesis and in human premalignant and malignant lesions. PhD Thesis, University of London
Goldstein DJ (1981) Errors in microdensitometry. Histochem J 13:251–267
Kugler P (1981) Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup. Histochemistry 71:433–449
Kugler P (1982) Aminopeptidase A is Angiotensinase A. I. Quantitative histochemical studies in the kidney glomerulus. Histochemistry 74:229–245
Pette D (1981) Microphotometric measurements of initial maximum reaction rates in quantitative enzyme histochemistry in situ. Histochem J 13:319–327
Pool Chr W, Diegenbach PC, Scholten G (1979) Quantitative succinate-dehydrogenase histochemistry. I. A methodological study on mammalian and fish muscle. Histochemistry 64:251–262
Rieder H, Teutsch HF, Sasse D (1978) NADP-dependent dehydrogenases in rat liver parenchyma. I. Methodological studies on the qualitative histochemistry of G6PDH, 6PGDH, malic enzyme and ICDH. Histochemistry 56:283–298
Robertson WR (1979) A quantitative cytochemical method for the demonstration of 5,3-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary. Histochemistry 59:271–285
Robertson WR, Davison B, Gandy M, Burdge R, Lambert A (1984) Computerized continuous monitoring of cytochemical enzyme reaction product formation by the Vickers M85A microdensitometer. Histochem J 16:651–656
Sinowatz F, Scheubeck M, Wrobel KH, Zwack M (1983) Histochemical localisation and quantification of glucose-6-phosphate dehydrogenase in bovine Leydig cells. Histochem J 15:831–844
Van Duijn P (1974) Fundamental aspects of enzyme cytochemistry In: Wisse E, Daems W Th, Molenaar I, Duijn P (eds) Electron microscopy and cytochemistry. North Holland, Amsterdam, pp 3–23
Van Noorden CJF, Tas J (1980) Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films. Histochem J 12:669–685
Van Noorden CJF, Butcher RG (1984) Histochemical localisation of NAD(P)-dependent dehydrogenase activity with four different tetrazolium salts. J Histochem Cytochem 32:998–1004
Van Noorden CJF, Tas J, Vogels IMC (1983) Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells. Histochem J 15:583–599
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Butcher, R.G. Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium; the effect of section thickness. Histochemistry 81, 567–572 (1984). https://doi.org/10.1007/BF00489536
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DOI: https://doi.org/10.1007/BF00489536