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The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

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Summary

A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3β-hydroxy-Δ5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500µmol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250µmol/l. The apparent Michaelis—Menten constant (K m) was 29µmol/l and Vmax 0.63µmol NAD+ reduced/min/cm3 corpora lutea.

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References

  • BADER, C. A., NASR, L. B., MONET, J., BACHELET, M., ASSAILLY, J. and ULMANN, A. (1984)In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdenisotometry.J. biol. Chem. 259, 11658–61.

    Google Scholar 

  • BASCH, R. S. & FINEGOLD, M. J. (1971) 3β hydroxysteroid dehydrogenase activity in the mitochondria of rat adrenal homogenates.Biochem. J. 125, 983–9.

    Google Scholar 

  • BUTCHER, R. G. (1978) The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions.Histochem. J. 10, 739–44.

    Google Scholar 

  • BUTCHER, R. G. (1984) Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium, the effect of section thickness.Histochemistry 81, 567–72.

    Google Scholar 

  • BUTCHER, R. G. & ALTMAN, F. P. (1973) Studies on the reduction of tetrazolium salts. II. The measurement of neotetrazolium chloride in tissue sections.Histochemistry 37, 351–63.

    Google Scholar 

  • CAFFREY, J. L., NETT, T. M., ABEL, J. H. & NISWENDER, G. D. (1979) Activity of 3β-hydroxy-Δ5-steroid dehydrogenase/isomerase in the ovine corpus luteum.Biol. Reprod. 20, 279–87.

    Google Scholar 

  • CHAYEN, J. (1984) ‘Quantitative Cytochemistry’, a precise form of cellular biochemistry.Biochem. Soc. Trans. 12, 887–98.

    Google Scholar 

  • CHAYEN, J., BITENSKY, L. & BUTCHER, R. G. (1973) The preparation of sections. InPractical Histochemistry, pp. 1–7. Chichester: John Wiley.

    Google Scholar 

  • CRABBE, M. J. C. (1982) An enzyme kinetics program for desk top computers.Comp. Biol. Med. 12, 263–83.

    Google Scholar 

  • DAVENPORT, G. R. & MALLETTE, L. E. (1966) Some biochemical properties of rabbit ovarian hydroxysteroid dehydrogenases.Endocrinology 78, 672–8.

    Google Scholar 

  • DIXON, M. & WEBB, E. C. (1979) Enzyme techniques—Measurement of velocity. InEnzymes (edited by DIXON, M. and WEBB, E. C.), pp. 7–9. London: Longman Group.

    Google Scholar 

  • GIBB, W. & HAGERMAN, D. D. (1976) The specificity of the 3β-hydroxysteroid dehydrogenase activity of bovine ovaries towards dehydroepiandosterone and pregnenolone: evidence for multiple enzymes.Steroids 28, 31–42.

    Google Scholar 

  • GORDON, M. T. & ROBERTSON, W. R. (1985) Is Δ5 3β hydroxysteroid dehydrogenase (Δ5 3βHSD) an allosteric enzyme?J. Endocr. Suppl.104, 105.

    Google Scholar 

  • KOWAL, J., FORCHIELLI, E. & DORFMAN, R. I. (1964) The Δ5-3β hydroxysteroid dehydrogenase of bovine corpus luteum and adrenal. I. properties, substrate specificity and cofactor requirements.Steroids 3, 531–44.

    Google Scholar 

  • KUGLER, P. (1981) Kinetic and morphometric measurements of enzyme reactions in tissue sections with new instrumental set-up.Histochemistry 71, 433–49.

    Google Scholar 

  • NEVILLE, A. M., ORR, J. C. & ENGEL, L. L. (1969) The Δ5 3β hydroxysteroid dehydrogenase activity of bovine adrenal microsomes.J. Endocrinol. 43, 599–609.

    Google Scholar 

  • NISWENDER, G. D., SAWYER, H. R., CHEN, T. T. & ENDRES, D. B. (1980). Action of luteinising hormone at the luteal cell level. In Advances in Sex Hormone Research (edited by Thomas, J. A. and Singh, R. L.) 4, 153–85. Urban and Schwarzenbera, Baltimore, Munich.

  • PETTE, D. (1981) Microphotometric measurement of initial maximum velocity rates in quantitative enzyme cytochemistryin situ.Histochem. J. 13, 319–27.

    Google Scholar 

  • PUPKIN, M., BRATT, H., WEISZ, J., LLOYD, C. W. & BALOGH, K. (1966). Dehydrogenases of the rat ovary. I. A histochemical study of Δ5, 3β and 20α hydroxysteroid dehydrogenases and enzymes of carbohydrate oxidation during the estrous cycle.Endocrinology 79, 316–27.

    Google Scholar 

  • ROBERTSON, W. R. (1979) A quantitative cytochemical method for the demonstration of Δ5, 3β hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary.Histochemistry 59, 271–85.

    Google Scholar 

  • ROBERTSON, W. R., DAVISON, B., GANDY, M., BURDGE, R. & LAMBERT, A. (1984) Computerised continuous monitoring of cytochemical enzyme reaction production formation by the Vickers M85A microdensitometer.Histochem. J. 16, 651–6.

    Google Scholar 

  • ROBERTSON, W. R., EARNSHAW, R. J. & LAMBERT, A. (1982a) The determination of the initial maximal velocity rate of rat ovarian 3β hydroxysteroid dehydrogenase activity.Histochemistry 74, 435–42.

    Google Scholar 

  • ROBERTSON, W. R., FROST, J., HOYER, P. E. & WEINKOVE, C. (1982b). 20α hydroxysteroid dehydrogenase activity in the rat corpus luteum, a quantitative cytochemical study.J. Steroid Biochem. 17, 237–43.

    Google Scholar 

  • STOWARD, P. J. (1980) Criteria for the validation of quantitative histochemical enzyme techniques. InTrends in Enzyme Histochemistry and Cytochemistry. Ciba Foundation Symposium 73 (New Series), pp. 11–31. Amsterdam: Excerpta Medica.

    Google Scholar 

  • YOSHIDA, N. (1979) Steroid specificity of human placental 5-ene-3β hydroxysteroid oxidoreductase.Steroids 33, 9–22.

    Google Scholar 

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  1. Department of Chemical Pathology, University of Manchester, Clinical Sciences Building, Hope Hospital, Eccles Old Road, M6 8HD, Salford, UK

    M. Gordon & W. R. Robertson

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  1. M. Gordon
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  2. W. R. Robertson
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Gordon, M., Robertson, W.R. The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary. Histochem J 18, 41–44 (1986). https://doi.org/10.1007/BF01676197

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  • DOI: https://doi.org/10.1007/BF01676197

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