(From the Division of Animal Physiology and Histochemistry, Department of Zoology, M.S. University of Baroda, Baroda, India) With i plate (fig.

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Histochemical demonstration of certain DPN-linked dehydrogenases and of aldolase in the red and white fibres of pigeon breast-muscle By J. C. GEORGE and C. L.

1 Histochemical demonstration of certain DPN-linked dehydrogenases and of aldolase in the red and white fibres of pigeon breast-muscle By J. C. GEORGE and C. L. TALESARA (From the Division of Animal Physiology and Histochemistry, Department of Zoology, M.S. University of Baroda, Baroda, India) With i plate (fig. i) Summary The distribution and localization-pattern of certain DPN-linked dehydrogenases (malic, lactic, D-glucose, glutamic, and a-glycerophosphate) were demonstrated histochemically in the red and white fibres of pigeon breast-muscle by using neotetrazolium as the hydrogen acceptor, under strictly anaerobic conditions. All the dehydrogenases studied showed distinctly higher enzyme activity in the narrow red fibres than in the broad white fibres. That of a-glycerophosphate was, however, found to be appreciably more abundant than other dehydrogenases in the broad fibres. A high concentration of aldolase, which forms an important link in the chain of enzymes in glycolysis, was histochemically demonstrated in the broad, white, glycogen-loaded fibres. Introduction DENNY-BROWN (1929), in a study of the red and white muscles of vertebrates, made some observations on the 'light' and 'dark' muscle-fibres in the breast-muscle of the pigeon. The later works on these two types of fibres have been reviewed by George and Naik (1957). More recently George and Naik (1958 a, b) have shown that the pectoralis major muscle of the pigeon consists of two distinct types of fibres, a broad, glycogen-loaded, white variety with few or no mitochondria, and a narrow, fat-loaded, red variety containing a large number of mitochondria. In a histochemical study of certain dehydrogenases in pigeon breast-muscle, George and Scaria (1958) could not detect the presence in the broad white fibres of any of the enzymes studied by the methods they employed. Later, however, it was possible to demonstrate the presence of succinic dehydrogenase and cytochrome oxidase in the broad white fibres by the use of improved histochemical methods (George and Talesara, 1961a). By employing an indirect method, certain oxidizing enzymes and a lipase were assessed quantitatively in the red and white fibres of this muscle. It was found that the broad white fibres also contain these enzymes, though in extremely low concentration (George and Talesara, 1961&). The present investigation was undertaken to demonstrate the localization and distribution-pattern of certain DPN-linked dehydrogenases (malic, lactic, D-glucose, a-glycerophosphate, and glutamic) in the red and white fibres of the pigeon breast-muscle, by improved histochemical methods. In addition, a histochemical study of [Quarterly Journal of Microscopical Science, Vol. 103, part, 1 pp , March 1962.]

42 George and Talesara Dehydrogenases and aldolase (which plays an important role in glycolysis) was also undertaken, so as to provide a comprehensive picture of the localization and distribution

2 42 George and Talesara Dehydrogenases and aldolase (which plays an important role in glycolysis) was also undertaken, so as to provide a comprehensive picture of the localization and distribution pattern of this enzyme in the two types of fibres. Material and methods The chemicals used as specific substrates for the various dehydrogenases were of 'C.P.' grade (Merck). Fructose i :6 diphosphate was obtained from L. Light & Co., and the cofactor, DPN, from Nutritional Biochemicals Corporation. DPN-linked dehydrogenases The literature on the various methods based on the use of tetrazolium salts for the histochemical demonstration of the different dehydrogenases has been recently reviewed by Pearse (i960) and by George and Talesara (1961a). Though nitro-blue tetrazolium with a high redox potential has been used in the most recent methods, in the present study neotetrazolium (NT) was used instead, because the nitro-blue compound was not available. For the histochemical demonstration of the various DPN-linked dehydrogenases the method as described by Pearse (1960), with NT as the hydrogen acceptor, was followed. NaCN was added into the incubation medium as an effective blocking agent for cytochrome oxidase and also as a trapping agent for any oxaloacetic acid that may be produced during the processing of the sections (Rosa and Velardo, 1954; George and Talesara, 1961a). Nicotinamide was also added in the incubation medium to prevent destruction of DPN (Mann and Quastel, 1941). The incubation medium used for the various dehydrogenases (in a total volume of 3 ml) was as follows: phosphate buffer (o-i M) at ph 7 i-o ml specific substrate, neutral salt (0-5 M) 0-6 ml neotetrazolium 2-0 mg DPN, 6 mg/ml 03 ml nicotinamide (o-i M) 0-3 ml MgCl 2 (0-05 M) 0-3 ml NaCN (0-03 M, adjusted to ph 7) 0-5 ml The pectoralis major muscle of normal, well-fed laboratory pigeons {Columba livid) was used throughout the study. For each set of experiments a bird was decapitated and bled. A piece of muscle 1 cm in length, consisting of the complete depth of the pectoralis major muscle, was immediately cut out. Fresh frozen sections, 20 to 40 fj. thick, were cut on a freezing microtome and floated on ice-cold phosphate buffer for nearly 10 min, so as to allow sufficient time for removal of the endogenous substrates before testing for the various dehydrogenases. The sections were transferred to the cuvettes containing the incubation mixtures with specific substrates, as described above, and incubated at 37 C for 10 to 30 min as required. In order to verify the specificity of the various DPN-linked dehydrogenases, a control was run side-by-side, with

3 aldolase in pigeon muscle 43 omission of the specific substrate from the incubation medium. After incubation the sections were washed thoroughly in phosphate buffer, fixed in 10% buffered neutral formalin at 4 0 C for 2 h., washed again, and mounted in glycerogel. The sections were immediately examined under a microscope and photographed. Aldolase This enzyme catalyses the splitting of fructose 1:6 diphosphate into 3- phosphoglyceraldehyde and a-phosphodihydroxyacetone. The only available method, that of Allen and Bourne (1943), was used for the histochemical demonstration of aldolase. Fructose 1:6 diphosphate was used as the substrate. Frozen sections of the muscle strip (fixed in 80% alcohol for 24 h) were incubated for 2 h at 37 C. Further enzymic breakdown of the two triosephosphates (the reaction products) was prevented by the presence of iodoacetate in the incubation medium, and the reaction products were precipitated by a mixture of magnesium and ammonium chlorides in alkaline solution. The precipitated phosphates were revealed by their conversion to the cobalt salt and finally to the brownish-black sulphide. An experiment in which the substrate was omitted was performed as a control. Results and discussion The size, localization, and distribution-pattern of all the various histochemically demonstrable DPN-linked dehydrogenases (fig. 1, A) other than of «-glycerophosphate, in the red as well as the white fibres of the pigeon breast-muscle, showed a more or less close resemblance to that of succinic dehydrogenase and cytochrome oxidase (George and Talesara, 1961a). The sarcoplasm of the fibres was stained slightly pink and contained sharp, bluishviolet diformazan granules, which were arranged in a definite linear fashion between the unstained myofibrils and thus showed some resemblance to the localization-pattern for mitochondrial staining. It is interesting to note that even in the case of lactic dehydrogenase, in the fresh frozen sections used in the present study as well as in the fixed sections obtained by the method of NovikofT and Masek (1958), the localization was intramitochondrial. The ph of the incubation medium was adjusted to 7 so as to avoid the possibility of getting a staining reaction in the absence of dehydrogenase, since the SH groups or glutathione may reduce exogenous DPN at alkaline ph levels (Zimmermann and Pearse, 1959). Tsao (i960) recently reported the electrophoretic heterogeneity of some of the dehydrogenases, but no differences of this sort can be studied histochemically. All the dehydrogenases studied showed a uniform distribution-pattern of enzyme activity in the individual fibre types throughout the depth of the muscle. (This muscle does not contain any intermediate types of fibres. Moreover, the diameter of the two types of fibres is more or less constant in the different regions and shows no significant variation from the average values (George and Naik, 1959).)

44 George and Talesara Dehydrogenases and All the DPN-linked dehydrogenases studied, except that of a-glycerophosphate, show a higher concentration of these enzymes in the narrow red fibres.

4 44 George and Talesara Dehydrogenases and All the DPN-linked dehydrogenases studied, except that of a-glycerophosphate, show a higher concentration of these enzymes in the narrow red fibres. The presence of a few diformazan granules in the broad white fibres suggests the presence of a few mitochondria and hence a low level of oxidative metabolism there. These histochemical results agree with the available biochemical data on the individual red and white fibres of this muscle (George and Talesara, 1961 b). However, it is rather surprising that lactic dehydrogenase, a glycolytic enzyme, shows a high level of activity in the narrow red fibres. Of the various dehydrogenases studied histochemically, the reduction of neotetrazolium was found to be almost as rapid in sections treated for malic dehydrogenase as in those treated for succinic dehydrogenase, in both types of fibres. Similar results were obtained in the biochemical study of this muscle (George and Talesara, 19616). It should be mentioned here that George and Scaria (1958) were unable to demonstrate the presence of D- glucose dehydrogenase histochemically in the breast-muscle of the pigeon, fl-glycerophosphate dehydrogenase was, however, appreciably more abundant in the broad fibre in comparison with other dehydrogenases present in the same fibre. It is rather difficult to define the exact localization and distributionpattern of a-glycerophosphate dehydrogenase in the two types of fibres because of the solubility of the enzyme, which results in diffused localization. Recently it has been shown that a-glycerophosphate is metabolized by two types of dehydrogenases, one a soluble DPN-linked enzyme (Sacktor and Cochran, 1957) and the other a structurally-bound dehydrogenase (Chance and Sacktor, 1958; Sacktor and Cochran, 1958) not requiring a nucleotide but reacting through the cytochrome system to oxygen. So far as the second type is concerned, it may not be present in the broad white fibres, since these fibres are poor in cytochromes. It should be noted here that a-glycerophosphate dehydrogenase plays a role in the metabolism of insect flight-muscles (Sacktor and Cochran, 1958). It is the first type that is the soluble DPN-linked enzyme which is present in considerable quantities in both the types of fibres. When the sections were incubated for nearly 30 min with the substrate but without any cofactor, no visible colour appeared in either of the fibres. These histochemical results on DPN-linked dehydrogenases, taken in conjunction with the earlier observations of George and Talesara (i960, 1961 a, b, c) and of Talesara (1961), suggest a high oxidative metabolism in the narrow red fibres and a low oxidative metabolism in the broad white fibres, and indicate that the broad white fibres of pigeon breast-muscle also possess mitochondria, though these may differ from those of the narrow fibres in size or form. The broad white fibres are therefore to be considered as fundamentally similar to the white fibres of other vertebrate skeletal muscles. FIG. 1 (plate). A, photomicrograph of a transverse section of the pectoralis major muscle of the pigeon, to show the localization and distribution-pattern of the various DPN-linked dehydrogenases in the narrow red and broad white fibres. B, photomicrograph of a transverse section of the pectoralis major muscle of the pigeon, to show the distribution-pattern of aldolase in the narrow red and broad white fibres.

5 Fio. i J. C. GEORGE and C. L. TALESARA

6 aldolase in pigeon muscle 45 The histochemical study of aldolase shows a higher activity of this enzyme in the broad white fibres (fig. 1, B). It is to be emphasized here that in a histochemical preparation, whenever a particular enzyme activity is found in appreciable amounts in both the types of fibres, it is very difficult to assess the relative concentrations in the two types. The types of fibre differ markedly in the density of the granular inclusions. The fat droplets present in the narrow type obliterate to some extent the true nature of the staining. The small diameter of the narrow fibres also makes interpretation difficult. It is clear that casual comparisons cannot be accurate. In this particular case the colour produced after the final reaction is dull brownish, which does not give a sharp contrast. Since aldolase is a soluble glycolytic enzyme, only the broad disposition was revealed: the exact intracellular localization could not be determined. Aldolase is reported to be present in the non-protein fraction from myofibrils of striated muscle (Perry and Zydowo, 1959), but, as was mentioned earlier, no definite differentiation in the localization of the enzyme in the myofibrils, sarcoplasm, or mitochondria was noticed. The suggestion of certain workers (Du Buy and Showacre, 1959; Showacre and Du Buy, 1959) that mitochondria in the intact cell contain all the glycolytic enzymes, does not seem to be convincing as far as the broad white fibres of pigeon breast-muscle or other vertebrate skeletal muscles rich in glycolytic enzymes are concerned, since the mitochondrial content of these fibres is always low as compared to the red fibres. It has been shown that during sustained muscular activity, pigeon breastmuscle utilizes fat as the chief fuel for energy (George and Jyoti, 1957). Hence a consideration of the fuel store in the two types of fibres (George and Naik, 1958a) and of phosphorylase activity (Dubowitz and Pearse, i960) suggests that the higher aldolase activity in the rapidly-contracting broad white fibres is an indication of the breakdown and synthesis of glycogen, in which these fibres are rich. On the other hand, the low aldolase activity in the slowlycontracting fatty, narrow, red fibres, which contain abundant mitochondria and the associated enzymes of the fatty acid oxidative system as well as those of the Krebs cycle, suggests that fat, in preference to glycogen, forms the chief fuel during sustained activity. References ALLEN, R. J. L., and BOURNE, G. H., J. exp. Biol., 20, 61. CHANCE, B., and SACKTOR, B., Arch. Biochem. Biophys., 76, 509. DENNY-BROWN, D., Proc. roy. Soc. Lond. B, 104, 371. DUBOWITZ, V., and PEARSE, A. G. E., i960. Nature, 185, 701. Du BUY, H. G., and SHOWACRE, J., J. Histochem. Cytochem., 7, 361. GEORGE, J. C, and JYOTI, D., J. Anim. Morph. Physiol., 4, 2. and NAIK, R. M., Ibid., 4, a, Nature, 181, Ibid., 181, Biol. Bull., n6, 239. and SCARIA, K. S., Quart. J. micr. Sci., 99, 469.

46 George and Talesara Dehydrogenases and aldolase in pigeon muscle GEORGE, J. C, and TALESARA, C. L., i960. Biol. Bull., 118, 262. 1961a. Quart. J. micr. Sci., 102, 131. 1961ft. J. cell. comp.

7 46 George and Talesara Dehydrogenases and aldolase in pigeon muscle GEORGE, J. C, and TALESARA, C. L., i960. Biol. Bull., 118, a. Quart. J. micr. Sci., 102, ft. J. cell. comp. Physiol. (in press). 1961c. J. Anim. Morph. Physiol. (in press). MANN, P. J. G., and QUASTEL, J. H., Biochem. J., 35, 502. NOVIKOFF, A. B., and MASEK, B., J. Histochem. Cytochem., 6, 217. PEARSE, A. G. E., i960. Histochemistry, theoretical and applied. London (Churchill). PERRY, S. V., and ZYDOWO, M., Biochem. J., 71, 220. ROSA, C. G., and VELARDO, J. T., J. Histochem. Cytochem., 2, no. SACKTOR, B., and COCHRAN, D. G., Biochem. Biophys. Acta, 25, Arch. Biochem. Biophys., 74, 266. SHOWACRE, J., and Du BUY, H. G., J. Histochem. Cytochem., 7, 370. TALESARA, C. L., J. Anim. Morph. Physiol. (in press). TSAO, M. U., i960. Arch. Biochem. Biophys., 86, 195. ZIMMERMANN, H., and PEARSE, A. G. E., J. Histochem. Cytochem., 7, 271.

Histochemical Observations on the Succinic Dehydrogenase and Cytochrome Oxidase Activity in Pigeon Breast-muscle. By J. C. GEORGE and C. L.

Histochemical Observations on the Succinic Dehydrogenase and Cytochrome Oxidase Activity in Pigeon Breast-muscle By J. C. GEORGE and C. L. TALESARA (From the Laboratories of Animal Physiology and Histochemistry,