Download 49 Localization of enzymes in certain secretory cells of Helix

49
Localization of e n z y m e s in certain secretory cells of
Helix tentacles
By NANCY J. LANE
(From the Cytological Laboratory, Department of Zoology, University Museum, Oxford.
Present address: Department of Pathology, Albert Einstein College of Medicine, Yeshiva
University, New York 61, N.Y., U.S.A.)
With 2 plates (figs. 3 and 4)
Summary
Secretory cells in the optic tentacles of the snails, Helix aspersa and H. pomatia, have
been investigated for the cytoplasmic localization of certain enzymes. The collar cells,
considered to be neurosecretory, and the lateral oval cells, were those examined. Acid
phosphatase activity is found in the cytoplasm of both cells, in scattered spheroids called
the/8-bodies. This enzymatic activity indicates that the /?-bodies may be lysosomes,
as does their ultrastructural appearance. In the 2 cell types, the activity of both alkaline
phosphatase and thiamine pyrophosphatase is localized in crescentic bodies considered
to correspond to the Golgi lamellae, and in some of the j8-bodies. The latter enzyme
also exists in the cortices of the ct-bodies which, like the /S-bodies, are lipid-containing
globules. The activity of both cytochrome oxidase and succinate dehydrogenase is
found, not only in granules, rods, and filaments interpreted as the mitochondria, but
also on the cortices of some or all of the /5-bodies. It is concluded that in invertebrates, the lipochondria may be the sites of activity of many different enzymes which
in vertebrates are restricted to distinct cell organelles.
Introduction
T H E cytological localization of certain enzymes in vertebrate tissues has been
well established. Investigations on vertebrate cells of various types have
shown that acid phosphatase activity is localized mainly in the lysosomes
(Novikoff, 1961), while thiamine pyrophosphatase (TPPase) is at highest
levels in the Golgi lamellae (Novikoff, Essner, Goldfischer, and Heus, 1962),
and cytochrome oxidase and succinate dehydrogenase are chiefly in the
mitochondria (Schneider, 1946; Kuff and others, 1956; Novikoff, 1961).
In invertebrate cells, little work has been done on intracellular enzymatic
localization. Although the mitochondrial enzymes appear to have a localization in the mitochondria similar to that in vertebrate cells (David, 1963a),
the distribution of the phosphatases does not exactly parallel that in vertebrate
tissues. Recent experiments, carried out to determine the sites of phosphatase
activity in invertebrate cells, have dealt with the neurones of Locusta (Lee,
1963), the spermatids in the ovotestis of Helix (Bradbury and Meek, 1963;
Meek and Bradbury, 1963), and the cerebral neurones of Helix (Lane, 1963;
Meek and Lane, 1963). These studies show that the phosphatases are localized in cytoplasmic components which may in some cases differ from those
which are the typical sites of phosphatase activity in vertebrate cells. In
Helix neurones, both acid phosphatase and TPPase are localized mainly
on the cortices of the lipochondria, i.e. the phospholipid and mixed lipid
[Quart. J. micr. Sci., Vol. 105, pt. 1, pp. 49-60, 1964.]
2421.1
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Lane—Enzymes in secretory cells of Helix tentacles
globules, as well as in the Golgi lamellae, while in Locusta neurones, the acid
phosphatase is present in smooth lamellar aggregates, and the TPPase activity
in membrane-bounded, spheroidal lipochondria, similar to the mixed lipid
globules of Helix. However, in the spermatid of Helix, alkaline phosphatase
and TPPase are localized in the Golgi lamellae; acid phosphatase is not present
in the spermatids at all, although some is found in the nurse cells. These
results indicate that the localization of each of these enzymes may differ from
one invertebrate group to another, as well as differing within the same animal,
without constant association with one particular cell organelle, as generally
occurs in the vertebrates. I wished to investigate further the localization of
the phosphatases in other, different cells of an invertebrate animal, such as
Helix, already examined for the enzyme content of some of its cells. Since
acid phosphatase and TPPase are found in the lysosomes and the Golgi
lamellae, both of which are considered to be concerned in secretory processes
(Novikoff and others, 1962), it seemed desirable to study the enzymatic
reactions in a cell involved in secretion. Neurosecretory 'collar' cells surround the ganglion in the optic tentacles of stylommatophoran pulmonates
(Lane, 1962). In the present study the collar cells in the snail Helix have been
tested for the presence of acid phosphatase (fig. 1), alkaline phosphatase, and
TPPase (fig. 2).
An investigation of the fine structure of other secretory cells, called the
lateral cells, in the optic tentacles of Helix (Lane, 1964), showed that their
cytoplasm contained a few electron-dense bodies similar to certain vertebrate
lysosomes. Since the marker enzyme of lysosomes is acid phosphatase, the
lateral cells were tested for the presence of this enzyme, and, in addition, in
view of their secretory nature, for the presence of TPPase and alkaline
phosphatase.
The tentacular secretory cells were also examined for the presence of the
mitochondrial enzymes, cytochrome oxidase and succinate dehydrogenase.
In light-microscopical examinations of the cytoplasmic inclusions of the
tentacular cells (Lane, 1962), routine techniques for mitochondria gave
uninterpretable results, because the large number of spheroidal granules in
the cell body obscured the sparse amount of cytoplasm that lay between them.
An ultrastructural study (Lane, 1964) showed that both collar and lateral
cells contained mitochondria. In this investigation I attempted to visualize
them by testing for the enzymes that have been found to be specifically
associated with the mitochondria of vertebrate cells, in order to determine
whether the same enzymes are also present in the mitochondria of these
tentacular cells.
Material and methods
The optic tentacles of the snails Helix aspersa Miiller and H. pomatia
Linnaeus were used as the material for this study. The tentacles of H.
pomatia were found to be preferable since they have fewer pigment cells
between the secretory cells than have those of H. aspersa; in the latter the
Lane—Enzymes in secretory cells of Helix tentacles
51
cells are sometimes obscured by the granules of the pigment cells that surround them. However, since the investigation into the histochemistry of the
inclusions of the secretory cells in the tentacles was carried out on H. aspersa
(Lane, 1962), both species were used in certain enzyme tests for the sake of
comparison.
The tissues were fixed in formaldehyde-calcium (F/Ca) (Baker, 1944)
either before or after incubation in the substrate media. In some cases the
tentacles were partially embedded in pieces of the spinal cord of the cat or
rabbit for support during sectioning, rapidly frozen in liquid nitrogen at
—1960 C, and sectioned at 4 ^ with a Cambridge rocking microtome in
a Slee cryostat. In others, sections were cut at i o / i o n a freezing microtome
without being embedded in spinal cord tissue. Each section was placed on
a coverslip or slide and dried briefly. Subsequently, after incubation, it was
mounted in Farrants's medium, glycerogel, or glycerine; in the case of the
last, the coverslips were ringed with varnish.
Alkaline phosphatase. The localization of the enzyme commonly known as
alkaline phosphatase (orthophosphoric monoester phosphohydrolase 3.1.3.1
(Anon., 1961)) •was determined by the method of Gomori (1952) with certain
modifications. The tentacular tissue was fixed for 3 h or overnight in F/Ca
and embedded in pieces of cat spinal cord fixed in the same way. Of the
frozen sections cut at 4 /x, those from H. pomatia were incubated for 1 \ h at
370 C, and those from H. aspersa for longer periods, 3 to 4 h at 370 C. The
reaction product was visualized with ammonium sulphide, and the sections
mounted in glycerogel. Control sections were incubated in the media without
substrate.
Acid phosphatase. Tests for the presence of acid phosphatase (orthophosphoric monoester phosphohydrolase 3.1.3.2) were carried out according to
Gomori's technique (1952) or by Holt's modification of it (1959). The tentacles were prepared in the same way as those tested for the activity of alkaline
phosphatase. With tissue from H. pomatia, incubation at 370 C proceeded
for 20, 30, or 40 min, with that from H. aspersa for 3 or 4 h. The final
reaction product was visualized by dilute ammonium sulphide. Control
sections were incubated in the substrate medium to which had been added an
inhibitor, io"3 M sodium fluoride.
Thiamine pyrophosphatase. Tests for the presence of TPPase were made
by the technique of Novikoff and Goldfischer (1961). The tentacles were
fixed, embedded, and sectioned as described for the alkaline phosphatase
test. The incubation medium was prepared as described by Novikoff and
Goldfischer, with a substrate (thiamine pyrophosphate) concentration of
0-03 M. Sections of the tentacles of H. pomatia were incubated for about
35 min at 370 C, but those of//, aspersa required incubation for 60 to 90 min.
The reaction product was visualized by means of ammonium sulphide, and
the sections were mounted in glycerogel. Control sections were incubated
in the substrate medium with o-i M uranyl nitrate as inhibitor.
Succinate dehydrogenase. The presence of succinate dehydrogenase
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Lane—Enzymes in secretory cells of Helix tentacles
(succinate: (acceptor) oxidoreductase 1.3.99.1) was tested for by David's
modification (1963ft) of the technique of Nachlas, Tsou, de Souza, Cheng,
and Seligman (1957). Unfixed tentacles of H. pomatia were embedded in
fresh rabbit spinal cord, rapidly frozen, and sectioned at 4 /x. The sections
were first placed in the substrate medium at 4° C for 30 min, to allow the
substrate to diffuse into the cells, and then incubated at 370 C for 10 min.
The incubation medium consisted of equal volumes of 0-2 M sodium
succinate and tris buffer (pH 7-0), to which were added small amounts of
vitamin K and ATP, and 1 drop of 0-5 M magnesium chloride to each ml
of medium; to this buffered substrate solution was added an equal volume
of o-i M nitro-BT. The sections were post-fixed in F/Ca, and mounted in
Farrants's medium. For the controls, 0-2 M sodium malonate was added to
the incubation medium as a competitive inhibitor.
Cytochrome oxidase. The localization of the enzyme cytochrome oxidase
(cytochrome C: O2 oxidoreductase 1.9.3.1) was determined by David's
modification (19636) of Burstone's procedure (1961). Unfixed tentacles of
H. pomatia were prepared as for the succinate dehydrogenase test, left in the
incubation medium at 40 C for 30 min, and incubated at 370 C for a further
30 min. The incubation medium consisted of 15 mg N-phenyl-p-phenylenediamine and 1 drop of 8-amino-i,2,3,4-tetrahydroquinoline dissolved in
0-5 ml ethanol, added to 25 ml of Krebs Ringer at pH 7-0. The sections were
post-fixed in 1 % cobalt acetate in F/Ca and mounted in Farrants's medium.
For the control sections, o-i M sodium azide was added to the normal incubation medium.
Results
The collar cells in Helix contain large quantities of spheroidal granules
which I have termed the a-bodies (Lane, 1964). So numerous are these
a-bodies that they almost obscure the cytoplasm lying between them; they
measure from 1 to 3 /x in diameter, and have been shown to contain some
phospholipid and perhaps cerebroside (Lane, 1962). Scattered at random
between them are other inclusions, called /J-bodies (Lane, 1964). Under
the electron microscope these (8-bodies are seen to contain varying numbers
of electron-dense granules which are sometimes clumped to one side of the
bounding membrane. Some of these enclosed electron-dense granules seem
to correspond to elementary neurosecretory granules (Lane, 1964). The
/J-body, in its mature form, measures about o-8 to 1 /x in diameter, and
appears to be formed from smaller, non-electron-dense spherical or kidneyshaped bodies by a process of growth and accumulation of electron-dense
material. Frequently, /^-bodies in diverse stages of development lie close
together within the perikaryon.
The lateral oval cells, at the ultrastructural level, contain electron-lucent
globules, which I shall also refer to as the a-bodies. As in the collar cells,
these are numerous and fill most of the cytoplasm. Dispersed between them
are bodies formed of electron-dense granules or rods; these bodies have a
Lane—Enzymes in secretory cells of Helix tentacles
53
fine structure very like that of some vertebrate lysosomes (Lane, 1964). I
shall refer to them as the /J-bodies.
Electron microscopical studies prove that both collar and lateral oval cells
contain Golgi complexes and mitochondria. However, no 'peri-nuclear
bodies', as described in light microscopical preparations (Lane, 1962), were
observed ultrastructurally. Similarly in this study, in no case were inclusions
identifiable as these peri-nuclear dictyosomes present in the cytoplasm of the
collar cells or the lateral oval cells.
-bodies
fi-bodies with
'internal satellites'
\Ofj
FIG. 1. Diagram of a section through a collar cell from an optic tentacle of H. asperm
showing acid phosphatase localized in the jS-bodies, some of which are aggregated in
one part of the cell.
Alkaline phosphatase. The distribution of alkaline phosphatase is the same
in both H. aspersa and H. pomatia. Its activity is not very intense in either
the collar cells or the lateral cells, and those cytoplasmic inclusions in which
it is present are not numerous. However, the collar cells do contain a small
number of scattered granules, less than 1 p in diameter, that display alkaline
phosphatase activity. A few filaments, 2 to 4 /x in length, and the cortices
of spheroids, 1 to 2 /JL in diameter, distributed at random through the cytoplasm, also show enzymatic activity. The spheroids are probably j8-bodies.
In the cytoplasm of the lateral oval cells, alkaline phosphatase is localized,
as in the collar cells, in a few scattered granules and filaments; occasionally
the latter partially surround certain of the a-bodies. Sometimes a lead reaction product, indicative of enzymatic activity, is found localized in a filament
that is V-shaped (fig. 4, c).
Control sections give no evidence of enzymatic activity in either species.
Acid phosphatase. In the cytoplasm of the collar cells in both species of
Helix, acid phosphatase activity is found in a few granules, less than 1 p in
diameter, dispersed at random in the perikaryon. The cortices of a smalL
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Lane—Enzymes in secretory cells of Helix tentacles
number of spheroids that measure from i to 2 JU., and which are scattered
throughout the cytoplasm with no particular distribution, also hydrolyse
/3-glycerophosphate at an acidic pH. These spheroids have the same size
range and distribution as the j8-bodies and occasionally they have 'internal
satellites' that also contain acid phosphatase activity. Often these spheroids,
or j8-bodies, with cortices rich in acid phosphatase, are aggregated together
in clumps that lie either to one side or at one end of the cells (figs. 1; 3, A).
The cytoplasm of the lateral oval cells in H. aspersa exhibits acid phosphatase activity distributed in the same way as it is in the collar cells (fig. 3, A, B).
However, in H. pomatia, spheroids with cortices rich in acid phosphatase are
nearly always scattered about as separate bodies, very rarely clumping together
in aggregations (fig. 3, D).
Sections incubated in the control media give a negative reaction.
Thiamine pyrophosphatase. The distribution of TPPase differs to a certain
extent in the two species of Helix.
In H. aspersa, the cytoplasm of the collar cells contains TPPase activity
that is localized in numerous scattered granules or rods, less than 1 n in
diameter, and in the cortices of a few spheroids agreeing in size with the
jS-bodies. Sometimes the cortices of the a-bodies also appear to possess
TPPase activity, but the reaction is usually very slight, and the lead reaction
product may only extend part of the way around the a-body; for this reason it
is difficult to determine whether the enzyme is truly on the cortices, or on
some element in the surrounding cytoplasm that is closely applied to their
surface.
The cytoplasm of the lateral cells in H. aspersa contains TPPase activity
localized on the cortices of a number of spheroids that are about 1 fx in
diameter; the cortices exhibit enzymatic activity either partially or completely
around their circumferences. Those spheroids that possess activity are quite
evenly spaced throughout the cytoplasm and are probably /9-bodies, as they
are not so numerous as the non-reactive a-bodies. The cytoplasm also contains filaments, 1 to 2 /x long, that exhibit TPPase activity; these have a
random distribution.
In H. pomatia the cytoplasm of the collar cells shows intense hydrolysis of
thiamine pyrophosphate occurring on the cortices of the ct-bodies and perhaps also of some /?-bodies (figs. 2; 3, c). However, not all of the cortices of
the a-bodies are positive, and some give a fainter reaction than others. In
some cases (figs. 2; 3, c), the reaction is so violent that it is impossible to
determine whether the enzyme is in fact restricted to the cortices. The
cortices rich in TPPase have a wide size range, from less than 1 to 3 /x.
They are not always spherical and may be elliptical or even almost rectangular.
Occasionally the TPPase is confined to one side of a spheroid; sometimes also
the reaction product assumes a U-shape around a spheroid, or the form of
granules lying against the spheroids (figs. 2; 3, c). In some cases filaments,
often in the form of crescents, are present and exhibit TPPase activity; these
lie between the a-bodies and are from 1 to 2 /JL in length.
Lane—Enzymes in secretory cells of Helix tentacles
55
The lateral oval cells in H. pomatia react to incubation for TPPase activity
in the same way as do the collar cells oiH. aspersa, i.e. the numerous a-bodies
or crescents applied to their surfaces give only a slight reaction, most of the
enzyme being localized in /9-bodies, granules, and rods.
The control sections of each species give negative results.
ft-bodies
granules in cpposition
to an cc -body
-ex-body
nicleus
cc-bodie.
crescents round
(X~ bodies
crescentic bodies
FIG. 2. Diagram showing the sites of thiamine pyrophosphatase in sections through
two collar cells from the optic tentacles of H. pomatia; one side of each cell contains so
many a-bodies displaying intense enzymatic activity, that the bodies appear united. It is
impossible to ascertain whether or not the activity is restricted to the cortices of these
a-bodies.
Succinate dehydrogenase. The collar cells of H. pomatia contain succinate
dehydrogenase that is localized in the cytoplasm in granules and rods found
in large numbers; these measure less than 1 /u. and are scattered evenly
throughout the cytoplasm. Sometimes activity is also present in thin filaments,
1 to 2 n long, and on the cortices of a few globules that measure from 1 to 2 /x
in diameter and are dispersed at random through the cytoplasm.
The lateral oval cells display succinate dehydrogenase localized in the same
way as in the cytoplasm of the collar cells.
Sections incubated in the control media to which sodium malonate had
been added show no enzymatic activity.
Cytockrome oxidase. In the cytoplasm of the collar cells of H. pomatia,
cytochrome oxidase is localized in numerous granules, rods, and filaments;
the granules and rods measure less than 1 /x in diameter, while the filaments
are 2 to 3 /x in length; they all are dispersed evenly through the perikaryon
(fig. 4, D). Some enzymatic activity is found on the cortices of spheroidal
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Lane—Enzymes in secretory cells of Helix tentacles
granules that range from less than 1 fj, to 2 /x (fig. 4, A, B). These are usually
scattered throughout the cytoplasm, but may be aggregated in clumps at one
end or side of the cell. There are indications that cytochrome oxidase is also
present in 'internal satellites' or inclusions lying within these spheroidal
granules (fig. 4, B).
Cytochrome oxidase is localized in the lateral oval cells in the same way as
in the collar cells.
Sections incubated in the control media with added sodium azide give
negative results.
Discussion
In the tentacular secretory cells of Helix, acid phosphatase is found to be
localized in the cortices of a number of spheroids and in granules. These
correspond in size and distribution to the /S-bodies and the smaller granular
inclusions that develop into jS-bodies. Under the electron microscope, the
structure of the /3-bodies is similar to that of some vertebrate lysosomes
(Lane, 1964): this similarity, in addition to the cytochemical evidence that
the jS-bodies contain acid phosphatase, the marker enzyme for lysosomes,
suggests that the j8-bodies correspond to lysosomes. Further, electron microscopical studies (Lane, 1964) showed that the /J-bodies are sometimes
aggregated together in the cytoplasm; this would correspond to the clumping
of the lysosomes in the light-microscopical acid-phosphatase preparations
described in this study. The occasional presence of 'internal satellites', rich
in acid phosphatase, in the /?-bodies or lysosomes no doubt corresponds to
the aggregations of electron-dense granules found ultrastructurally inside
some of the larger /?-bodies (Lane, 1964).
The electron-dense granules within the /?-bodies have been found in some
cases to correspond in size and in structure to the elementary neurosecretory
granules (Lane, 1964). Association of acid phosphatase activity with
neurosecretory granules has been observed in neurones elsewhere. In vertebrate hypothalamo-hypophysial systems, it has been shown that the amount
of acid phosphatase not only increases with the increased synthesis of aldehydeFIG. 3 (plate). All the cells shown in this figure are from the optic tentacles of Helix and
were fixed in formaldehyde/calcium. Frozen sections were cut at 10 /x before incubation for
enzymatic activity.
A, collar cell from H. aspersa showing acid phosphatase localized in the /3-bodies (jS) in the
form of scattered granules and spheroids. Note the accumulation of jS-bodies in the cytoplasm
at one side of the nucleus (n).
B, lateral oval cell from H. aspersa, showing acid phosphatase localized in the jS-bodies (j8).
Note the aggregation of j3-bodies at one end of the cell and the 'internal satellites' (sat)
within them, n, nucleus.
c, collar cell from H. pomatia (compare with fig. 2) after incubating for thiamine pyrophosphatase; note the sites of the enzyme, which are the cortices of the ce-bodies (a) and
/3-bodies, granules (gr) and crescents (cr), the last two often lying around cv-bodies. cl, clumps
of a-bodies, with cortices containing the enzyme.
D, lateral oval cell from H. pomatia, exhibiting acid phosphatase activity in scattered
spheroidal and granular j3-bodies (j3).
FIG.
3
N. J. LANE
FIG. 4
N. J. LANE
Lane—Enzymes in secretory cells of Helix tentacles
57
fuchsin-positive material in the neurosecretory cells (Kobayashi and Farner,
i960), but also parallels hormonal production (Sobel, 1961). Electron
microscopical examinations of the neurosecretory granules of vertebrate
hypothalamic, cerebellar, and cord neurones (Novikoff, 1962; Novikoff and
others, 1962) show accumulation of reaction product when incubated for acid
phosphatase. Further, it is to be noted that the electron-lucent vesicles at
the ends of the Golgi lamellae are considered to correspond to lysosomes,
since they have been shown ultrastructurally to contain acid phosphatase
(Novikoff and others, 1962); these vesicles are formed in the same way as the
electron-dense elementary granules, by budding and vesiculation from the
ends of the Golgi lamellae (Dalton, i960; Palay, i960; Bern and others, 1961
and 1962; Scharrer and Brown, 1961; Murakami, 1962; Lederis, 1962,
Rohlich, Aros, and Vigh, 1963), so that the latter might reasonably be considered lysosomal as well.
Histochemical tests showed (Lane, 1962) that 'dispersed lipid droplets'
were present in the collar and lateral oval cells. It seems probable that the
j8-bodies or lysosomes and the Golgi complexes correspond ultrastructurally
to these 'lipid droplets' (Lane, 1964). In the cerebral neurones of H. aspersa,
acid phosphatase is localized in the lipochondria, both the 'blue' (phospholipid) and yellow (mixed lipid) globules, which are considered to correspond
to lysosomes (Lane, 1963; Meek and Lane, 1963); in certain vertebrate
neural cells, the lipochondria have also been shown to contain acid phosphatase
and are said to be lysosomes (Koenig, 1962; Ogawa and others, 1961). Hence,
in a number of cases, lipid globules have been found to be equivalent to the
lysosomes, and the tentacular secretory cells seem to be no exception.
In Helix tentacular cells, the localization of alkaline phosphatase in filaments and U-shaped forms suggests that some of the enzyme may be situated
in the smooth lamellae of the Golgi complex. The larger V-shaped forms,
up to 4 fi, in length, would no doubt correspond to two Golgi regions lying at
right angles to one another, as has been observed to occur in electron microscopical studies (Lane, 1964). Alkaline phosphatase associated with the
FIG. 4 (plate). All the cells shown in this figure are from the optic tentacles of Helix and
were fixed in formaldehyde/calcium.
A, collar cell from H. pomatia, cut at 4 fa and post-fixed, incubated for cytochrome oxidase,
which is localized in the mitochondria (m); note the enzymatic activity on the cortices of some
^-bodies (j3), aggregated in one part of the cell at x. n, nucleus.
B, H. pomatia collar cell, post-fixed after being sectioned at 4 fj. and incubated for the
activity of cytochrome oxidase. Note the sites of activity in the mitochondria (?«) and the
cortices of the |8-bodies (j3); note also the 'internal satellites' (sat) in some ^-bodies which
are grouped together at x. n, nucleus.
C, lateral oval cell of H. aspersa,fixedand sectioned at 4 fi before incubation for alkaline
phosphatase, which is present in granules (gr) and filaments ( / ) in the cytoplasm round
the nucleus (n).
D, collar cell from H. pomatia, sectioned at 4 JJ. and post-fixed after incubation for cytochrome oxidase, which is chiefly localized in the mitochondria (m), but also in some /3-bodies.
(/3). The central cell (c) is the same one as in B, but at a different focus. Cells incubated for
succinate dehydrogenase have the same appearance.
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Lane—Enzymes in secretory cells of Helix tentacles
Golgi apparatus has been previously observed in the spermatid of Helix
(Bradbury and Meek, 1963), and in certain vertebrate cells (Deane and
Dempsey, 1945; Bourne, 1951; Novikoff, Korson, and Spater, 1952;
Schneider and Kuff, 1954). The occasional presence of alkaline phosphatase
activity in the form of granules and on spheroids suggests that, as in the
cerebral neurones of H. aspersa, its activity is also found in some lipidcontaining bodies, in this case probably the /3-bodies.
In the collar and lateral oval cells of Helix, TPPase is often localized in
filaments or crescents, which suggests that it is situated in the Golgi lamellae,
as it is in vertebrate cells (Novikoff and others, 1962). However, thiamine
pyrophosphate is also hydrolysed on the cortices of some of the a-bodies and
^-bodies. Hence, TPPase seems to be present both in the Golgi lamellae
and in the lipid globules, as in the cerebral neurones of H. aspersa (Meek and
Lane, 1963). In some cases, though, notably the collar cells of H. pomatia,
nearly all the TPPase appears to be on the cortices of the a- and /3-bodies.
In these instances enzymatic activity may also be present in the Golgi,
though only in one lamella of a group, which would be impossible to resolve
at the level of light microscopy. Such a situation occurs in the neurones of
H. aspersa: under the light microscope, most of the TPPase is localized in the
cortices of certain lipid globules (Lane, 1963); under the electron microscope,
one or more Golgi lamellae of a group may contain the enzyme (Meek and
Lane, 1963).
The tests carried out for the mitochondrial enzymes show that the mitochondria in the tentacular secretory cells of Helix, present in the form of
granules, rods, and filaments, contain the same enzymes as do the mitochondria of vertebrate cells. Both cytochrome oxidase and succinate dehydrogenase
sometimes also occur on the cortices of globules that are often aggregated at
one end of the cell and which occasionally possess 'internal satellites'. This
suggests that these are the jS-bodies that also display acid phosphatase activity,
in other words, the lysosomes. However, the group of enzymes that is
typically associated with the lysosome in vertebrate cells does not include
succinate dehydrogenase, while cytochrome oxidase is said to be completely
absent (Novikoff, 1961). It has recently been shown in the cerebral neurones
of H. pomatia (David, 1963a) that cytochrome oxidase and succinate dehydrogenase are localized not only in the mitochondria, but also on the cortical
borders of some of the lipid globules. It has already been mentioned that the
lipid globules in the neurones of H. aspersa contain acid phosphatase (Lane,
1963; Meek and Lane, 1963), so that Helix cerebral neurones provide another
instance, in addition to that described here in the tentacular cells, of 'lysosomal' lipochondria exhibiting the activity of enzymes that are typically
mitochondrial.
The association of various different enzymes with the lipid globules suggests
that the lipochondria of invertebrate cells generally, and the lysosomal
jS-bodies of the tentacular cells in particular, may play an important role in
the functioning of the cell.
Lane—Enzymes in secretory cells of Helix tentacles
59
The intracellular localization of enzymes in invertebrates appears to differ
somewhat from that in vertebrates. In vertebrate cells, as far as is known,
particular enzymes are generally restricted to certain distinct cytoplasmic
components. In invertebrates this restriction does not hold good. Further,
the localization of a particular enzyme is different in some cases from that of
the same enzyme in vertebrate cells. Indeed, certain cell organelles, the
lipochondria, are the sites of activity of many different enzymes. Thus in
invertebrates, particular enzymes cannot always be used as 'markers' for any
particular cell organelle.
I would like to thank Dr. G. B. David and Mr. A. W. Brown for their
invaluable help with technique and photography in certain of the tests
performed. I am grateful to Dr. J. R. Baker, F.R.S., for his supervision and
encouragement during the course of this work, and to Professor J. W. S.
Pringle, F.R.S., for accommodation in his Department. I am indebted to the
Canadian Federation of University Women for financial assistance in the form
of their Travelling Fellowship.
References
Anon., 1961. In Report of the commission on enzymes of the international union of biochemistry.
Oxford (Pergamon Press).
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