Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1966 Apr 1;29(1):113–128. doi: 10.1083/jcb.29.1.113

CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

H Dariush Fahimi 1, Morris J Karnovsky 1
PMCID: PMC2106951  PMID: 4288329

Abstract

The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BRODY I. A., ENGEL W. K. EFFECTS OF PHENAZINE METHOSULFATE IN HISTOCHEMISTRY. J Histochem Cytochem. 1964 Dec;12:928–929. doi: 10.1177/12.12.928. [DOI] [PubMed] [Google Scholar]
  2. CASCARANO J., ZWEIFACH B. W. Factors influencing the histochemical demonstration of coenzyme-dependent dehydrogenases and diaphorases. J Biophys Biochem Cytol. 1959 Mar 25;5(2):309–318. doi: 10.1083/jcb.5.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DAWSON D. M., GOODFRIEND T. L., KAPLAN N. O. LACTIC DEHYDROGENASES: FUNCTIONS OF THE TWO TYPES RATES OF SYNTHESIS OF THE TWO MAJOR FORMS CAN BE CORRELATED WITH METABOLIC DIFFERENTIATION. Science. 1964 Feb 28;143(3609):929–933. doi: 10.1126/science.143.3609.929. [DOI] [PubMed] [Google Scholar]
  4. DE DUVE C., WATTIAUX R., BAUDHUIN P. Distribution of enzymes between subcellular fractions in animal tissues. Adv Enzymol Relat Subj Biochem. 1962;24:291–358. doi: 10.1002/9780470124888.ch6. [DOI] [PubMed] [Google Scholar]
  5. DUBOWITZ V., PEARSE A. G. A comparative histochemical study of oxidative enzyme and phosphorylase activity in skeletal muscle. Z Zellforch Microsk Anat Histochem. 1960;2:105–117. doi: 10.1007/BF00744575. [DOI] [PubMed] [Google Scholar]
  6. FARBER E., STERNBERG W. H., DUNLAP C. E. Histochemical localization of specific oxidative enzymes. I. Tetrazolium stains for diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J Histochem Cytochem. 1956 May;4(3):254–265. doi: 10.1177/4.3.254. [DOI] [PubMed] [Google Scholar]
  7. FASSKE E., GERLACH U., STEINS I., THEMANN H. DEHYDROGENASEN-DARSTELLUNG IM ELEKTRONENMIKROSKOPISCHEN ZELLBILD. Z Naturforsch B. 1964 Oct;19:887–892. [PubMed] [Google Scholar]
  8. FRANZINI-ARMSTRONG C., PORTER K. R. SARCOLEMMAL INVAGINATIONS CONSTITUTING THE T SYSTEM IN FISH MUSCLE FIBERS. J Cell Biol. 1964 Sep;22:675–696. doi: 10.1083/jcb.22.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Graymore C. 'Nothing' dehydrogenase in the retina. Nature. 1965 Jun 26;206(991):1360–1361. doi: 10.1038/2061360a0. [DOI] [PubMed] [Google Scholar]
  10. HIMMELHOCH S. R., KARNOVSKY M. J. The histochemical demonstration of glyceraldehyde-3-phosphate dehydrogenase activity. J Biophys Biochem Cytol. 1961 Mar;9:573–581. doi: 10.1083/jcb.9.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KARNOVSKY M. J. Simple methods for "staining with lead" at high pH in electron microscopy. J Biophys Biochem Cytol. 1961 Dec;11:729–732. doi: 10.1083/jcb.11.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. KURTZ S. M. A new method for embedding tissues in Vestopal W. J Ultrastruct Res. 1961 Oct;5:468–469. doi: 10.1016/s0022-5320(61)80020-8. [DOI] [PubMed] [Google Scholar]
  13. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LePAGE G. A., SCHNEIDER W. C. Centrifugal fractionation of glycolytic enzymes in tissue homogenates. J Biol Chem. 1948 Dec;176(3):1021–1027. [PubMed] [Google Scholar]
  15. MARGRETH A., MUSCATELLO U., ANDERSSON-CEDERGREN E. A MORPHOLOGI- CAL AND BIOCHEMICAL STUDY ON THE REGULATION OF CARBOHYDRATE METABOLISM IN THE MUSCLE CELL. Exp Cell Res. 1963 Dec;32:484–509. doi: 10.1016/0014-4827(63)90189-7. [DOI] [PubMed] [Google Scholar]
  16. MATHISEN J. S., MELLGREN S. I. SOME OBSERVATIONS CONCERNING THE ROLE OF PHENAZINE METHOSULFATE IN HISTOCHEMICAL DEHYDROGENASE METHODS. J Histochem Cytochem. 1965 May-Jun;13:408–409. doi: 10.1177/13.5.408. [DOI] [PubMed] [Google Scholar]
  17. MUSCATELLO U., ANDERSSON-CEDERGREN E., AZZONE G. F., von der DECKEN The sarcotubular system of frog skeletal muscle. A morphological and biochemical study. J Biophys Biochem Cytol. 1961 Aug;10(4):201–218. doi: 10.1083/jcb.10.4.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Markert C. L., Møller F. MULTIPLE FORMS OF ENZYMES: TISSUE, ONTOGENETIC, AND SPECIES SPECIFIC PATTERNS. Proc Natl Acad Sci U S A. 1959 May;45(5):753–763. doi: 10.1073/pnas.45.5.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. NEILANDS J. B. Studies on lactic dehydrogenase of heart. III. Action of inhibitors. J Biol Chem. 1954 May;208(1):225–230. [PubMed] [Google Scholar]
  20. Ogawa K., Barrnett R. J. Electron cytochemical studies of succinic dehydrogenase and dihydronicotinamide-adenine dinucleotide diaphorase activities. J Ultrastruct Res. 1965 Jun;12(5):488–508. doi: 10.1016/s0022-5320(65)80043-0. [DOI] [PubMed] [Google Scholar]
  21. PAIGEN K., WENNER C. E. The intracellular location of the glycolytic dehydrogenases in liver and hepatoma. Arch Biochem Biophys. 1962 Apr;97:213–216. doi: 10.1016/0003-9861(62)90067-x. [DOI] [PubMed] [Google Scholar]
  22. PEARSE A. G., HESS R. Substantivity and other factors responsible for formazan patterns in dehydrogenase histochemistry. Experientia. 1961 Mar 15;17:136–141. doi: 10.1007/BF02160833. [DOI] [PubMed] [Google Scholar]
  23. PETTE D., BRANDAU H. Intracellular localization of glycolytic enzymes in cros-striated muscles of Locusta migratoria. Biochem Biophys Res Commun. 1962 Oct 31;9:367–370. doi: 10.1016/0006-291x(62)90056-6. [DOI] [PubMed] [Google Scholar]
  24. PETTE D., BUECHER T. [Proportionally constant groups in relation to the differentiation of enzyme activity patterns of skeletal muscles in rabbits]. Hoppe Seylers Z Physiol Chem. 1963 Mar;331:180–195. doi: 10.1515/bchm2.1963.331.1.180. [DOI] [PubMed] [Google Scholar]
  25. PORTER K. R., FRANZINI-ARMSTRONG C. THE SARCOPLASMIC RETICULUM. Sci Am. 1965 Mar;212:72–81. doi: 10.1038/scientificamerican0365-72. [DOI] [PubMed] [Google Scholar]
  26. ROBERTSON J. D., BODENHEIMER T. S., STAGE D. E. THE ULTRASTRUCTURE OF MAUTHNER CELL SYNAPSES AND NODES IN GOLDFISH BRAINS. J Cell Biol. 1963 Oct;19:159–199. doi: 10.1083/jcb.19.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. ROSA C. G., TSOU K. C. DEMONSTRATION OF THE SJOESTRAND MEMBRANE PARTICLES BY THE ELECTRON CYTOCHEMICAL METHOD. Nature. 1965 Apr 3;206:103–105. doi: 10.1038/206103a0. [DOI] [PubMed] [Google Scholar]
  28. SCHOLLMEYER P., KLINGENBERG M. [On the cytochrome content of animal tissue]. Biochem Z. 1962;335:426–439. [PubMed] [Google Scholar]
  29. SMITH B. THE LOCALIZATION OF ENZYMES WITHIN SKELETAL MUSCLE FIBERS USING THE TETRAZOLIUM TECHNIQUE. J Histochem Cytochem. 1964 Nov;12:847–851. doi: 10.1177/12.11.847. [DOI] [PubMed] [Google Scholar]
  30. STEIN J. M., PADYKULA H. A. Histochemical classification of individual skeletal muscle fibers of the rat. Am J Anat. 1962 Mar;110:103–123. doi: 10.1002/aja.1001100203. [DOI] [PubMed] [Google Scholar]
  31. Shaw C. R., Koen A. L. On the identity of "nothing dehydrogenase". J Histochem Cytochem. 1965 Jul-Aug;13(6):431–433. doi: 10.1177/13.6.431. [DOI] [PubMed] [Google Scholar]
  32. WATSON M. L. Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol. 1958 Jul 25;4(4):475–478. doi: 10.1083/jcb.4.4.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. ZIMMERMANN H., PEARSE A. G. Limitations in the histochemical demonstration of pyridine nucleotide-linked dehydrogenases (nothing dehydrogenase). J Histochem Cytochem. 1959 Jul;7(4):271–275. doi: 10.1177/7.4.271. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES