SOME EXPERIMENTS ON THE MICROSCOPICAL DEMONSTRATION
OF ZYMOHEXASE IN ANIMAL TISSUES
BY R. J. L. ALLEN AND G. H. BOURNE,* From the Low Temperature Station for Research
in Biochemistry and Biophysics, University of Cambridge and Department of Scientific
and Industrial Research, Cambridge and the University Laboratory of Physiology, Oxford
(Received 27 June 1943)
(With Plate 2)
The presence in extracts of animal tissues and yeast
of an enzyme catalysing the cleavage of hexosediphosphate (fructofuranose-i :6-diphosphate) into
two molecules of triosephosphate was demonstrated
by Meyerhof & Lohmann (1934a):
the most recent Joyet-Lavergne (1938), have regarded
mitochondria as centres of cellular respiration, but
the evidence adduced to support this conception is
very alight. In the present work attempts have been
made to demonstrate zymohexase microscopically in
CH,O.PO,H,
CH.OPO.H,
C=O
CH.OH
CH.O.PO.H,
fructofuranose-1:6-diphotphate
CHO
+
CH(OH)
CH.O.PO.H,
dihydroxyacetone- phosphoglyceraldehyde
phosphate
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 (19346) 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 ^>arts of the cell.
Many workers, the first being Kingsbury (1912) and
• MacKenzie-MacKinnon Research Fellow of the
Royal College of Physicians of London and the Royal
Qollege of Surgeons of England.
the cells of various animal tissues, in the hope of
throwing some light on the question of localization
of respiration.
METHOD
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 hexosediphbsphate and triosephosphate are not precipitated from aqueous
solution by magnesia mixture (magnesium chloride
and ammonium hydroxide), whereas inorganic phos-
62
R. J. L. ALLEN and G. H. BOURNE
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 370 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.
Reagents used
Sodium hexosediphosphate, 4 % solution. This was
prepared by treating calcium hexosediphosphate
with sodium oxalate.
Magnesia mixture (Kursanov's
(1938)
formula).
5-5 g. MgCl,.6H,0 and 7 g. NH,C1 were dissolved
in 35 ml. $N NHiOH, let stand 1 hr., and filtered.
60 ml. of 4.N NH,OH were then added.
Purified sodium hexosediphosphate solution.
For-
mula 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,
o-iM
solution'.
i-86 g. of
iodoacetic acid were neutralized to bromothymol
blue with N NaOH and diluted to 100 ml.
Sodium fluoride, o-iM solution.
Cobaltous chloride, CoCl,.6H,O, 2 % solution.
Ammonium sulphide solution. Freshly prepared by
diluting i ml. of the concentrated light yellow
tion (prepared as directed by Treadwell &
1930) with about 50 ml. distilled water.
The substrate mixture
Three substrate mixtures were used:
A. 10 ml. purified sodium hexosediphosphate
(formula I), 5 ml. HiO, 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.
RESULTS
Experiment 1
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 560 C. Sections (10 p) were
floated on to the slides with warm water, dried at
370 C. f6r 24hr. and stored at 30 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 littledarker than the controls (see below). With
the trachea there appSeared 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.
Experiment 2
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.
Microscopical demonstration of zymohexase in animal tissues
Skeletal muscle. 1 hr.: as heart muscle. Even
raer oil immersion individual fibres showed a
homogeneous brown appearance; there appeared to
be no partition of brown material between fibrillae
and sarcoplasm (PI. 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 CQiwective
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.
Experiment 3
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 (PI. 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
PI. 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.
Experiment 4
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
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). Tn 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.
DISCUSSION
The strong browning of skeletal muscle with the
present technique is in accordance with the wellknown 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
R. J. L. ALLEN and G. H. BQURNE
zymohexase i9 usually considered to be associated
with the more soluble parts of the muscle tissue.
This result may, however, be due to the limitations
of microsxx>pical 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 contains1 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 wa"5 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 incubatj
with the more concentrated substrate mixtures,
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.
SUMMARY
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 OF
less uniformly throughout the cell.
REFERENCES
KURSANOV, A. (1938). Biokhimiya, 3, 467.
BOURNE, G. H. (1943). Quart. J. Exp. Phytiol. 3a, 1.
GOMORJ, G. (1941). J. Cdl. Comp. Pkytiol. 17, 71.
HERBERT,
D.,
GORDON,
H.,
SUBRAHMANYAN,
V.
MEYHRHOF, O. & LOHMANN, K. (1934 a).
&
GREEN, D. E. (1940). Biochem. J. 34, 1108.
JOYET-LAVERGNE, P. (1938). Rev. gin. Sci. pur. appl.
31 Jan., p. 1.
KINGSBURY, B. F. (1912). Anat. Rec. 6, 39.
Biochem. Z.
37i, 89-
MEYERHOF, O. & LOHMANN, K. (19346). Biochem. 2.
373, 413TREADWELL, F. P. & HALL, W. T. (1930). Analytical
Chemistry, 1. New York: Wiley and Sons.
EXPLANATION OF PLATE 2
Fig. 1. Section of jejunum of rat, showing dark staining
of the smooth muscle layer and no reaction in the villi.
x8o.
Fig. 2. Skeletal muscle of rat. Showing uniform dark
coloration of the fibres, x 800.
Fig. 3. Liver of rat. Showing diffuse darkening of cells
around the central vein. The participate black material
in the section is extraneous precipitate deposited on top
of the section, x 40.
JOURNAL OF EXPERIMENTAL BIOLOGY, 20, 1
PLATE 2
Fig. 1
Fig. 2
Central
vein
Fig- 3
ALLEN AND BOURNE—SOME EXPERIMENTS ON THE MICROSCOPICAL DEMONSTRATION
OF ZYMOHEXASE IN ANIMAL TISSUES (pp. 61-64)