Some Experiments on the Microscopical Demonstration of Zymohexase in Animal Tissues

The reaction, catalysed by zymohexase, as the enzyme was called, is now generally regarded as occupying a central position in the complex series of changes by which carbohydrate is broken down in living tissues.

Meyerhof & Lohmann (1934b) found zymohexase to be present in extracts of various tissues of the rabbit, frog, goose and mouse, but the concentration of the enzyme was much greater in skeletal muscle of the rabbit than in any other tissue examined.

Although it is probable that in the living cell oxidation of carbohydrate can proceed in more than one way, it seems certain that the oxidation of triosephosphate and of pyruvate derived from it is a reaction of great importance in respiration. Therefore zymohexase, which is essential for the formation of these substances from hexose, must be regarded as of fundamental importance in cellular respiration.

Cytologists have long endeavoured to localize respiratory activities in certain parts of the cell. Many workers, the first being Kingsbury (1912) and the most recent Joyet-Lavergne (1938), have regarded mitochondria as centres of cellular respiration, but the evidence adduced to support this conception is very slight. In the present work attempts have been made to demonstrate zymohexase microscopically in the cells of various animal tissues, in the hope of throwing some light on the question of localization of respiration.

The technique was based On the following points :

1. Zymohexase is remarkably active in alkaline solution ; even at pH 11 it retains about 50 % of its activity at its optimal pH of about 9 (Herbert, Gordon, Subrahmanyan & Green, 1940).

2. Further enzymic breakdown (oxidation) of triosephosphate formed under the influence of zymohexase can be prevented by the presence of lodoacetate.

3. Inorganic phosphate is rapidly liberated from triosephosphate in alkaline solution at room temperature, whereas hexosediphosphate is stable under these conditions (Meyerhof & Lohmann, 1934a, b).

4. Except in the presence of a large excess of inorganic phosphate hexosediphosphate and triosephosphate are not precipitated from aqueous solution by magnesia mixture (magnesium chloride and ammonium hydroxide), whereas inorganic phos-phate is rapidly thrown down as magnesium ammonium phosphate.

5. Magnesium ammonium phosphate and cobaltous chloride form an insoluble complex, the cobalt in which can be converted to brownish black cobaltous sulphide, which is visible under the microscope, by treatment with ammonium sulphide after the method of Gomori (1941).

6. Although Herbert et al. (1940) found that preparations of zymohexase from rabbit muscle were inactivated by contact with organic solvents, preliminary experiments indicated that intact tissues can be fixed in alcohol or acetone without serious loss of their zymohexase activity.

Sections of fresh frog and rat tissue and of alcohol-and acetone-fixed tissues of the rat and guinea-pig were incubated at 37 °in one of a number of substrate mixtures (see below) for various periods. They were then removed, washed in many changes of diluted magnesia mixture (5 ml. mixture to 100 ml. water) for varying times (on one occasion for 18 hr.) to remove any hexosediphosphate absorbed by the tissues, placed in a 2% solution of cobaltous chloride for 5 min., washed in at least three changes of distilled water for a total time of 15 min., placed in ammonium sulphide solution for 5 min., washed in distilled water, dehydrated, cleared and mounted in balsam.

It was expected that the following sequence of changes would take place in the tissues during incubation: (1) formation of triosephosphate from hexosediphosphate in the presence of zymohexase, (2) liberation of inorganic phosphate from triosephosphate in the presence of OH’ ions (from the ammonia in the magnesia mixture), (3) precipitation of inorganic phosphate as magnesium ammonium phosphate.

Sodium hexosediphosphate, 4% solution. This was prepared by treating calcium hexosediphosphate with sodium oxalate.

Magnesia mixture (Kursanov’s (1938) (formula). 5 ·5 g. MgCl₂.6H₂O and 7 g. NH₄Cl were dissolved in 35 ml. 5N NH₄OH, let stand 1 hr., and filtered. 60 ml. of 4N NH₄OH were then added.

Purified sodium hexosediphosphate solution. Formula I: 40 ml. 4% sodium hexosediphosphate solution and 20 ml. magnesia mixture were mixed, let stand 30 min. and the precipitated inorganic phosphate present as impurity filtered off. Formula II: 20 ml. 4% sodium hexosediphosphate, 20 ml. distilled water and 20 ml. magnesia mixture were mixed and treated as for formula I.

Sodium iodoacetate, 0 ·1 M solution. 1 ·86 g. of iodoacetic acid were neutralized to bromothymol blue with N NaOH and diluted to 100 ml.

Sodium fluoride, 0 ·1 M solution.

Cobaltous chloride, CoCl₂.6H₂O, 2% solution.

Ammonium sulphide solution. Freshly prepared by diluting 1 ml. of the concentrated light yellow solution (prepared as directed by Treadwell & hall 1930) with about 50 ml. distilled water.

Three substrate mixtures were used :

• A. 10 ml. purified sodium hexosediphosphate (formula I), 5 ml. H₂O, 1 ·7 ml. sodium iodoacetate.

• B. 10 ml. purified sodium hexosediphosphate (formula I) 3 ·3 ml. water, 1 ·7 ml. sodium fluoride, 1 ·7 ml. sodium iodoacetate.

• C. 15 ml. purified sodium hexosediphosphate (formula II), 7 ·5 ml. water, 2 ·5 ml. sodium iodoacetate.

Sodium fluoride was added to B to prevent hydrolysis of hexosediphosphate under the influence of phosphatase. It was subsequently found unnecessary since under the conditions used the tissues showed no phosphatase activity.

Skeletal muscle, spinal cord and trachea of a 3 weeks’ old rat were fixed for 24 hr. in 80 % alcohol, passed through absolute alcohol to xylene (12 hr.) and embedded in wax at 56 ° C. Sections (10 p) were floated on to the slides with warm water, dried at 37 ° C. for 24hr. and stored at 3 °C. for 3 weeks. Before use they were passed through xylene and alcohol to water. The sections of muscle were incubated for 30 min. in substrate mixture A, the sections of spinal cord and trachea for the same time in substrate mixture B.

Following treatment with cobaltous chloride and ammonium sulphide the muscle was found to be very little darker than the controls (see below). With the trachea there appeared to be an aggregation of dark particulate matter especially located round the cartilage cells; this reaction was probably spurious. The spinal cord gave practically no reaction.

It was thought that the negative reaction with these tissues was probably due to inactivation of zymohexase during the pre-treatment, which included embedding in wax. In the subsequent experiments the sections were not embedded but cut on the freezing microtome.

Heart muscle, skeletal muscle, small intestine, brain, liver, kidney, skin, lung, rectum, salivary gland and stomach of a week old rat were fixed in 80 % alcohol for 24 hr. at room temperature, washed in water for 5 –10 min., frozen and sectioned, and the sections dropped into substrate mixture A. Sections were removed and examined after 1 and 2 hr.

Heart muscle. 1 hr. : browned diffusely. 2 hr. : slight browning in most sections, stronger browning in patches.

Skeletal muscle. I hr. : as heart muscle. Even under oil immersion individual fibres showed a homogeneous brown appearance; there appeared to be no partition of brown material between fibrillae and sarcoplasm (Pl. 2, fig. 2).

Small intestine. 1 hr. : an irregular dark precipitate formed over the surface of the section, which was itself browned diffusely. 2 hr. : precipitate formed over most of section, strong browning of muscular coat, epithelium also darkened.

Brain. 1 hr. : very faint browning. Cerebellum and cerebrum stained with the same intensity. In the cerebellum the Purkinje cells were darker than other elements. In general all the cells seemed to be slightly darker than the surrounding fibres. Under oil immersion the cytoplasm of the cerebellar cells appeared to contain large dark circular bodies of which there were more than one to a cell. 2 hr. : more intense reaction, distributed as before.

Liver, 1 hr. : light browning. The surface of the section was covered with dark precipitate. 2 hr. : same as at 1 hr.

Kidney. 2 hr. : some dark precipitate, some browning, which was more intense in some glomeruli than in the tubules.

Skin, 1 hr. : slight browning. Cells of hair follicles slightly darker than other parts of the section. 2 hr. : same as 1 hr.

Lung, 1 hr. : no reaction. 2 hr. : no reaction.

Rectum. 1 hr. : light browning of smooth muscle coat and slight browning of submucous connective tissues. 2 hr. : same as at 1 hr. except that slight browning of some deeper epithelial cells.

Salivary gland. 1 hr. : no reaction.

Stomach. 1 hr. : light browning of smooth muscle coat and submucous connective tissue near the muscle ; no reaction in epithelium. 2 hr. : same as at 1 hr.

It is believed that the browning observed in the various tissues in this experiment was due to the activity of zymohexase.

In this experiment substrate mixture C, containing a lower concentration of hexosediphosphate than A or B, was used. The extraneous dark precipitate formed over the surface of some of the sections in Exp. 2 was absent.

Sections of some of the tissues of the same animal used for Exp. 2 were used. The procedure was as in Exp. 2 but in some cases (indicated below) washing after incubation and before cobalt treatment was continued for much longer than usual.

Kidney. 1 hr. : general light browning, intense immediately beneath capsule. Some small areas of the section showed very dark staining.

Intestine. 1 hr. : slight browning ; smooth muscle coat darker than mucosa (Pl. 2, fig. 1).

Liver, 1 hr. : moderate browning. There was a gradation in the intensity of staining, the reaction being faintest with the peripheral cells of the lobules and increasing progressively in intensity as the central veins of the lobules were approached (see Pl. 2, fig. 3). 3 hr. : washed overnight before cobalt treatment. Moderate browning. With this section large numbers of lilac-coloured crystals were observed on the surface of the tissue after cobalt treatment but before sulphide treatment. The nature and cause of this curious artefact is unknown.

Skeletal muscle. 2 hr. : very dark diffuse browning. 3 hr. : washed overnight before cobalt treatment. Same as at 2 hr.

Brain. 2 hr. : moderate browning of both fibres and cells. 3 hr. : washed overnight before cobalt treatment. Browner than at 2 hr.

Pieces of liver, brain, skeletal muscle and adrenal from a 3 weeks old rat were treated as in Exp. 3 except that they were fixed in absolute acetone instead of alcohol. They were incubated for various times in substrate mixture C.

Liver, 1 hr. : slight browning and slight extraneous dark precipitate. 4 hr. : much darker than at 1 hr.

Brain. 1 hr. : slight browning and slight extraneous black precipitate. 4 hr. : much darker than at 1 hr.

Skeletal muscle. 1 hr. : moderate browning.

Adrenal. 4 hr. :slight browning. Slight extraneous dark precipitate.

Controls of two types were used. First, sections of all the specimens used were passed through the cobalt chloride and ammonium sulphide reagents in order to demonstrate any preformed inorganic phosphate in the tissues (Gomori, 1941). In all cases no reaction was obtained. Secondly, sections of all the specimens used were treated by Gomori’s (1941) method for demonstrating alkaline phosphatase. The appearance of these sections (see Bourne, 1943) was so different from the zymohexase preparations as to exclude the possibility of the present results being due to alkaline phosphatase activity.

The strong browning of skeletal muscle with the present technique is in accordance with the well-known fact that this tissue is especially rich in zymohexase. This also affords important additional evidence that the reactions obtained were not due to. alkaline phosphatase, because skeletal muscle contains very little of this enzyme as compared with, for example, kidney. The positive results with heart muscle were also to be expected.

Transverse sections of the skeletal muscle preparations when examined under an oil immersion objective showed no difference in intensity of reaction between the fibrils and the sarcoplasm ; this is perhaps surprising in view of the fact that zymohexase is usually considered to be associated with the more soluble parts of the muscle tissue. This result may, however, be due to the limitations of microscopical resolution, or to diffusion of some of the reagents.

In all the preparations from the alimentary tract the smooth muscle coat reacted more intensely than any other part of the section. This suggests that smooth muscle contains appreciable amounts of zymohexase, an observation which does not appear to have been previously recorded.

In the cells of all organs which gave a positive reaction the brown colour was diffused throughout the cell and did not appear to be localized in any particular part of the cell. Certain cells in the brain were exceptional in containing darker staining bodies in the cytoplasm, but even here the rest of the cells stained a dark brown as well. These results suggest (assuming they are not due to diffusion of the reagents and we believe they are not) that the respiratory activity of the cells, at least as regards the metabolism of carbohydrate, is not a property of any particular cell element but is a property of both the nuclei and cytoplasm as a whole.

The presence of an extraneous dark precipitate covering the surface of some sections after incubation with the more concentrated substrate mixtures, and subsequent treatment, is difficult to explain. It occurred independently of whether the sections showed browning or not and, therefore, its occurrence does not invalidate the results obtained, but its presence indicates that the technique requires further investigation before it can be recommended for general use.

A technique is described for the microscopical demonstration of zymohexase (an important enzyme in the breakdown of carbohydrate in living tissues).

The technique involves incubating sections of tissue in a substrate mixture containing hexosediphosphate in the presence of magnesia mixture and iodoacetate, followed by treatment of the magnesium ammonium phosphate so formed with cobaltous chloride and ammonium sulphide.

The enzyme was found by this method to be present in skeletal, heart and smooth muscle. In cellular organs it was found to be diffused more or less uniformly throughout the cell.