Summary
Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread on object glasses before mounting the sections to be incubated. In this way, the auxiliary enzymes were interposed between glass slide and section thus preventing loss of formazan generated within the sections. Creatine kinase activity was found to be localized in finely dispersed form along the myofibrils and as large granules in the sarcoplasm of myocardium and skeletal muscle. The formazan produced specifically by creatine kinase (test minus control), as measured cytophotometrically at 585 nm, was completely inhibited by 2mm 2,4-dinitrofluorobenzene, a specific inhibitor of creatine kinase activity. The control reaction was unaffected by the inhibitor. The results obtained with the present method are similar to results obtained with the far more complicated semipermeable membrane technique. The introduction of auxiliary enzymes in the polyvinyl alcohol method enables the development of histochemical methods for many enzymes by linking the reactions to a dehydrogenase reaction.
Similar content being viewed by others
References
ALTMAN, F. P. (1976) Tetrazolium salts and formazans.Progr. Histochem. Cytochem. 9/3 1–56.
ALTMAN, F. P. & CHAYEN, J. (1965) Retention of nitrogenous material in unfixed sections during incubations for histochemical demonstration of enzymes.Nature 207 1205–6.
BUTCHER, R. G. (1971) Tissue stabilisation during histochemical reactions: The use of collagen polypeptides.Histochemie 28 231–5.
BUTCHER, R. G. (1981) The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions.Histochem. J. 10 739–44.
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.
CAIN, D. F. & DAVIES, R. E. (1962) Breakdown of adenosine triphosphate during a single contraction of working muscle.Biochem. Biophys. Res. Commun. 8 361–6.
DE VRIES, G. P. & MEIJER, A. E. F. H. (1976) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. VId-Glucose-phosphate isomerase and phosphoglucomutase.Histochemistry 50 1–8.
FAHIMI, H. D. & AMARASINGHAM, C. R. (1964) Cytochemical localization of lactic dehydrogenase in white skeletal muscle.J. Cell Biol. 22 29–48.
FREDERIKS, W. M., MARX, F., JONGES, G. N. & VAN NOORDEN, C. J. F. (1987a) Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique.J. Histochem. Cytochem. 35 175–80.
FREDERIKS, W. M., MARX, F. & VAN NOORDEN, C. J. F. (1987b) Quantitative histochemical assessment of heterogeneity of glycogen phosphorylase activity in liver parenchyma from fasted rats using the semipermeable membrane technique and the PAS-reaction.Histochem. J. 19 150–6.
FREDERIKS, W. M., MARX, F. & STROOTMAN, F. (1987c) Demonstration of creatine kinase in myocardial and skeletal muscle using the semipermeable membrane technique.J. Mol. Cell. Cardiol. 19 311–17.
HENDERSON, B. (1984) Quantitative cytochemistry of lactate dehydrogenase activity.Cell Biochem. Function. 2 149–52.
HÜTHER, G. & LUPPA, H. (1977) Demonstration of acetylcholinesterase by semipermeable membrane technique: estimation of soluble and fixation-labile portions in different regions of the central nervous system.Histochemistry 51 245–51.
INFANTE, A. A. & DAVIES, R. E. (1965) The effect of 2,4-dinitrofluorobenzene on the activity of striated muscle.J. Biol. Chem. 240 3996–4001.
LEVITSKY, D. O., LEVCHENKO, T. S., SAKS, V. A., SHAROV, V. G. & SMIRNOV V. N. (1978) The role of creatine phosphokinase in supplying energy for the calcium pump system of heart sarcoplasmic reticulum.Membrane Biochem. 2 81–96.
LOJDA, Z., GOSSRAU, R. & SCHIEBLER, T. H. (eds) (1979)Enzyme Histochemistry. A Laboratory Manual, pp. 216–23. Berlin, Heidelberg, New York: Springer Verlag.
MCMILLAN, P. J. (1967) Different demonstration of muscle and heart type lactic dehydrogenase of rat muscle and kidney.J. Histochem. Cytochem. 15 21–31.
MEIJER, A. E. F. H. (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I Acid phosphatase.Histochemie 30 31–9.
MEIJER, A. E. F. H. (1978) Adaptations of methods in enzyme histochemistry to the different metabolic conditions in cells and tissues.Cell. Mol. Biol. 23 159–70.
MEIJER, A. E. F. H. (1985) The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique.Histochem. J. 17 1271–5.
MEIJER, A. E. F. H. & DE VRIES, G. P. (1974) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue section. IV Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating).Histochemistry 40 349–59.
MEIJER, A. E. F. H. & DE VRIES, G. P. (1975) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).Histochemistry 43 225–36.
MEIJER, A. E. F. H. & STEGEHUIS, F. (1980) Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles.Histochemistry 66 75–91.
PETTE, D. & BRANDAU, H. (1966) Enzym-Histogramme and Enzymaktivitätsmuster der Leber. Nachweis Pyridinnukleotidspezifischer Dehydrogenasen im Gelschichtverfahren.Enzymol. Biol. Clin. 6 79–122.
ROBERTSON, W. R., DAVIDSON, B., GANDY, M., BURDGE, R. & LAMBERT, A. (1984) Computerized continuous monitoring of cytochemical enzyme reaction product formation by the Vickers M 85A microdensitometer.Histochem. J. 16 651–6.
SCHOLTE, J. R. (1973) On the triple localization of creatine kinase in heart and skeletal muscle cells of the rat: evidence of the existence of myofibrillar and mitochondrial isoenzymes.Biochim. Biophys. Acta 305 413–27.
SJÖVALL, K. (1967) A tetrazolium technique for the histochemical localization of ATP:creatine phosphotransferase.Histochemie 10 336–40.
STOWARD, P. J. (1980) Criteria for the validation of quantitative histochemical enzyme techniques.Excerpta Medica Ciba Found. Symp. 73 11–31.
STOWARD, P. J., CAMPBELL, J. P. & AL-SARRAJ, B. (1982) Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle. IV. A post-coupling technique.Histochemistry 74 367–77.
VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1984) Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.J. Histochem. Cytochem. 32 998–1004.
VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1986a) The "out of range" error in microdensitometry.Histochem. J. 18 397–8.
VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1986b) A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity.Histochem. J. 18 364–70.
VAN NOORDEN, C. J. F., KOOY, A., VOGELS, I. M. C. & FREDERIKS, W. M. (1985) On the nature of the "nothing dehydrogenase" reaction.Histochem J. 17 1111–18.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Frederiks, W.M., Marx, F. & Van Noorden, C.J.F. Histochemical demonstration of creatine kinase activity using polyvinyl alcohol and auxiliary enzymes. Histochem J 19, 529–532 (1987). https://doi.org/10.1007/BF01687359
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01687359