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AMER. ZOOL., 24:187-197 (1984)
Enzymology of Plasma Membranes of Insect Intestinal Cells'
MICHAEL G. WOLFERSBERGER
Department of Biology, Temple University,
Philadelphia, Pennsylvania 19122
SYNOPSIS. The enzymology of insect intestinal cell plasma membranes is a field of scientific research that is in the earliest stages of development. In this paper the few published
studies specifically designed to isolate plasma membranes from insect intestinal cells and
determine the enzymes associated with them are reviewed in light of both older studies
that approached these problems less directly and recent results from our laboratory.
In the past few years reliable methods have been developed for the isolation of specific
portions of plasma membranes from the epithelial cells of the midguts of a few insect
larvae. These membrane preparations have been assayed for a variety of enzyme activities.
Alkaline phosphatase, leucine aminopeptidase and 7-glutamyl transpeptidase have shown
promise as potential markers for the plasma membranes of insect larval midgut cells.
However, only the latter enzyme currently stands unchallenged as a marker for the apical
portion of the plasma membrane of insect midgut columnar epithelial cells. No enzymes
can yet be considered to be even tentatively established as markers for the basal or lateral
portions of insect intestinal cells.
INTRODUCTION
Much of the exciting current research
on vertebrate transporting epithelia is of a
biochemical nature. Such studies are possible because methods have been developed for isolation, in a functional state, of
specific portions of the plasma membrane
of vertebrate epithelial cells and enzymes
characteristic of each isolated portion of
the cell membrane have been established.
Studies of some arthropod transporting
epithelia have progressed to the stage
where answers to the most interesting
questions will be best obtained using biochemical techniques (Hanrahan and Phillips, 1983; Harvey et ai, 19836; Towle,
1984).
When Dr. Towle invited me to participate in this symposium he suggested that
I address the topic of "Enzymology of
Membrane Fractions from Insect Intestinal Cells." I viewed this suggestion as an
opportunity to assess the current status of
the development of methods for isolation
of plasma membranes from insect intestinal cells and identification of enzyme
markers for different portions of the isolated cell membranes. I screened the mate-
rial reviewed in this paper from references
resulting from a search of the recent literature dealing with insects plus physiology and biochemistry of the digestive system plus physiological studies involving
enzymes. I relied on references cited in
recent papers plus texts and review articles
as a means of locating pertinent work published more than five years ago. Since this
review is concerned with plasma membrane bound enzymes of insect intestinal
cells I have omitted many excellent studies
of enzymes found in the lumen of insect
digestive tracts as well as those concerned
with enzymes of insect tissues that are not
usually considered part of the digestive
tract. This screening process left me with
a comparatively short reference list but one
that none the less should provide the reader
access to virtually all studies pertinent to
the very young and limited field reviewed.
GENERAL BACKGROUND
The essential features of the alimentary
system of a primitive insect larva are illustrated in Figure 1. At the simplest level the
insect digestive tract can be subdivided into
foregut, midgut and hindgut (Wigglesworth, 1972). The foregut begins at the
mouth, includes the buccal cavity (into
1
From the Symposium on Cellular Mechanisms of which the salivary glands empty), the
Ion Regulation in Arthropods presented at the Annual
Meeting of the American Society of Zoologists, 2 7 - esophagus, and the crop; it ends with the
proventriculus, a valve structure of varying
30 December 1982, at Louisville, Kentucky.
187
188
MICHAEL G. WOLFERSBERGER
complexity separating the foregut from the
midgut. The foregut is of ectodermal origin and its epithelial cells are covered by
cuticle. The midgut may be only a simple
tube (ventriculus) or it may be a tube with
blind sacs (gastric caeca) branching from FIG. 1. Diagram of the digestive tract of an insect.
its anterior end (Fig. 2). It is frequently A, salivary gland; B, pharynx; C, crop; D, provenobserved that cells in different regions of triculus; E, midgut; F, Malpighian tubules; G, small
the midgut differ morphologically and intestine; H, rectum. Diagram modified from Wigappear to be specialized for either secre- glesworth (1972).
tion or absorption (Cioffi, 1984); this specialization is observed even in the case of
midguts with nominally only one cell type site of digestion and nutrient absorption in
(Ferreira et al., 1981). The midgut is of detritivores, in other insects the hindgut is
endodermal origin and the apical plasma generally belived to be specialized primarmembrane of its epithelial cells is not pro- ily for water reabsorption. The mechanism
tected from the contents of the midgut of water reabsorption by insect hindgut is
lumen by cuticle. However, the midgut reviewed in another paper of this sympolumen is usually lined by an acellular peri- sium (Hanrahan, 1984).
trophic membrane which is secreted by
Over the years many experiments have
midgut cells. The peritrophic membrane attempted to discover how a variety of
serves as both a mechanically protective foodstuffs are digested and absorbed by
cover over the epithelial cells and a diffu- insects. A general picture has emerged; it
sion barrier between the cells and the mid- appears to hold for many members of the
gut contents (Terra and Ferreira, 1981). select group of insects that have been studThe midgut appears to be the primary site ied in the laboratory which are not nutriof digestion and nutrient absorption in most tionally dependent on symbiotic micoorinsects studied that are not dependent upon ganisms (Wigglesworth, 1972; House,
symbiotic microorganisms for primary 1974). However, because of the great
digestion of ingested food (House, 1974). dietary diversity among insects there are
Additionally, it is an important organ of bound to be numerous exceptions.
ion regulation in at least some insects (HarIngested food in the mouth is mixed with
vey, 1982). In the region of the sphincter
fluid
from the salivary glands; this fluid
separating the midgut from the hindgut
often
contains one or more enzymes of carMalpighian tubules branch off from the
bohydrate
digestion. This mixture is then
digestive tract. Although they are anatomsubjected
to
varying amounts of mechanically continuous with the digestive tract,
ical
breakdown
and enzymatic digestion,
Malpighian tubules generally appear to be
highly specialized for ion regulation and by saliva enzymes, in the foregut. The forethus are usually considered as being part gut contents are released in a controlled
of an insect's excretory system (Ramsay, manner into the midgut where the vast
1958). Although Malpighian tubules are majority of enzymatic digestion takes place.
generally disregarded in discussions of Most enzymes of carbohydrate digestion
insect digestion and absorption, there is and possibly all enzymes of lipid and proevidence that just as the midgut plays a role tein digestion are produced by the cells of
in insect ion regulation, the Malpighian the midgut. Many of these digestive
tubules play a role in nutrient absorption enzymes appear to be secreted by the cells
and even digestion in some insects (Wig- and are found mixed with the contents of
glesworth, 1972). Distal to the midgut is the midgut lumen. However, some digesthe hindgut which includes the rectum and tive enzymes remain bound either to the
terminates with the anus. The hindgut is epithelial cell membranes or within the
of ectodermal origin and the cells are cov- epithelial cells. The monosaccharides,
ered by cuticle. Although it is the primary amino acids, fatty acids, short peptides, and
simple lipids resulting from enzymatic
ENZYMES OF INSECT CELL MEMBRANES
digestion of foodstuffs in the midgut are
absorbed into the cells of the midgut epithelium. Some nutrients which remain in
the gut contents when they pass out of the
midgut are absorbed along with water and
salts in the hindgut; a few may be absorbed
from the gut fluid that enters the Malpighian tubules; and the remainder pass
out of the insect in its feces.
PRELIMINARY STUDIES
One of the most comprehensive studies
designed to determine which of the
enzymes found in the digestive tract of an
insect are secreted into the lumen and
which of these enzymes remain associated
with the cells was carried out by Terra et
al. (1979) on the larvae of the fly Rhynchosciara americana. The midgut of this
insect larva is illustrated in Figure 2. They
removed the entire midguts from hundreds
of larvae. Some of the whole midguts,
including contents, were homogenized
whereas others were further dissected, as
follows. The gastric caeca were removed
and their contents were collected; the ventriculus was opened and the peritrophic
membrane, including contents, was
removed;finallythe ventriculus was cut into
anterior and posterior halves. This dissection yielded five separate fractions of midgut: endoperitrophic contents (from the
peritrophic membrane and its contents),
ectoperitrophic contents (from the fluid in
the gastric caeca), caecal cells, anterior
ventriculus cells, and posterior ventriculus
cells. A homogenate was prepared of each
fraction and aliquots of homogenates of
whole midgut as well as those of each fraction were assayed for various enzyme activities. Some of the results of these assays are
shown in Table 1.
The specific activity of a few enzymes
such as cellulase is similar in all midgut
fractions while the specific activities of
numerous other enzymes—lactase, maltase, /3-glucosidase, a- and /3-galactosidase,
glycylglycine dipeptidase, carboxypeptidases A and B, carboxylesterase, lipase, and
both acid and alkaline phosphatases—are
at least three times greater in the homogenates of midgut cells than in the homogenates of midgut contents. However, only
189
FIG. 2. Diagram of the larval midgut of theflyRhynchosaara americana. AV, anterior ventriculus; EC,
ectoperitrophic space; EN, endoperitrophic space; GC,
gastric caecum; PM, peritrophic membrane; PV, posterior ventriculus. Diagram modified from Ferreira
etal. (1981).
three of these enzyme activities—acid phosophatase, alkaline phosphatase, and carboxylesterase—are restricted exclusively
to midgut cells. Carboxylesterase activity
is further restricted to the cells of only the
anterior ventriculus and the gastric caeca.
Although some lipase activity was found in
endoperitrophic midgut contents, this
enzymic activity is almost entirely localized
in the cells of the anterior ventriculus where
it shows the greatest enrichment of specific
activity of any of the enzymes assayed. This
localization of lipase activity is consistent
with the results of Treherne (1958) who
showed that in Periplaneta americana most
triglyceride hydrolysis and essentially all
fatty acid absorption occurred in the anterior portion of the midgut.
The specific activities of several
enzymes—a-amylase, carboxypeptidase A,
chymotrypsin, trehalase, and trypsin—are
appreciably greater in homogenates of
midgut contents than in homogenates of
the midgut tissue. However, of these
enzyme activities only those of chymotrypsin and trypsin appear to be restricted to
the extracellular midgut contents where,
like a-amylase and cellulase activities, they
are distributed almost equally between the
endoperitrophic and ectoperitrophic fluids.
The enrichment of trypsin and chymotrypsin activities in R. americana midgut
contents may have been expected on the
basis of other studies which invariably
found such enzyme activities in the lumen
of insect digestive tracts (Law et al., 1977).
Similarly the enrichment of a-amylase
activity in R. americana midgut contents
might have been expected since this enzyme
190
MICHAEL G. WOLFERSBERGER
TABLE 1.
Specific activities of digestive enzymes in homogenates of whole midguts and various portions of midguts from
Rhynchosciara americana larvae.
Specific enzyme activity
Enzyme activity
assayed
Lactase
Maltase
Sucrase
Trehalase
a-Amylase
a-Glucosidase
/3-Glucosidase
a-Galactosidase
0-Galactosidase
Cellulase
Arginine
aminopeptidase
Leucine
aminopeptidase
Glycylglycine
dipeptidase
CarboxypeptidaseA
CarboxypeptidaseB
Chymotrypsin
Trypsin
Carboxylesterase
Lipase
Acid phosphatase
Alkaline
phosphatase
Venlnculus cells
Midgul contents
Whole midgul
Endoperiirophic
Ectopentrophic
2.7
10.5
7.2
66.0
63.0
17.7
24.8
144.0
345.0
150.0
—
—
—
Caeca cells
12.6
30.0
15.1
26.7
23.1
31.0
43.9
2.0
15.4
14.1
15.2
98.8
16.2
42.7
13.8
41.6
30.5
38.4
21.0
1.5
6.6
0.6
0.0
56.7
308.0
208.0
36.1
257.0
14.8
12.4
32.1
146.0
38.2
108.0
456.0
1,348.0
1,257.0
188.0
600.0
67.4
16.3
59.4
115.0
29.0
36.4
91.6
53.9
121.0
90.4
2.0
2.1
212.0
17.1
105.0
6.4
502.0
43.9
141.0
16.2
486.0
34.4
638.0
61.8
0.0
7.1
0.0
0.0
2.5
4.9
0.0
2.8
0.0
552.0
528.0
202.0
351.0
1.6
4.2
0.0
0.0
0.0
0.0
0.0
0.0
7.6
0.2
35.7
35.2
301.0
56.9
98.5
1,398.0
12.5
0.0
7.9
19.7
6.3
1.5
0.4
0.0
3.8
11.3
Data from Terra et al. (1979). Specific activities are expressed in mUnits/mg protein; —, activity not
reported.
has been shown to be present in the saliva
of other insects (Chippendale, 1978) and
thus would enter the midgut along with the
contents of the foregut. However, the
enrichment of a-amylase activity in the cells
of/?, americana anterior midgut favors production of at least a portion of this enzyme
by midgut cells. The nearly equal distribution of specific activity between the
endoperitrophic and ectoperitrophic fluids
does not hold for trehalase, arginine and
leucine aminopeptidases, carboxypeptidase A, or glycylglycine dipeptiase all of
which show much higher specific activities
in the ectoperitrophic fluid than in the
endoperitrophic fluid, an indication that
the peritrophic membrane is a barrier to
the diffusion of these enzyme proteins. The
finding that trehalase activity is enriched
in the midgut contents but present in the
cells is consistent with the results of other
studies (Wyatt, 1967) that have found both
soluble and membrane bound trehalase
activities to be widely distributed in insects.
The distribution of peptidase activities in
R. americana midgut fractions may have
been somewhat less expected since in three
species of hematophagous insects Glossina
morsitans (Gooding and Rolseth, 1976),
Rhodnius prolixus, and Triatoma phyllosoma
(Houseman and Downe, 1980) carboxypeptidase activity was found predominately in the contents of the digestive tract
but aminopeptidase activities were essentially restricted to the cells.
The results presented in Table 1 in conjunction with the other studies mentioned
above provide some interesting information about the distribution of digestive
enzyme activities in the midgut of/?, americana as well as some other insects. They
effectively eliminate enzymes such as chymotrypsin and trypsin while suggesting
enzymes such as carboxylesterase and alkaline phosphatase as possible candidates for
membrane markers. However, in most
191
ENZYMES OF INSECT CELL MEMBRANES
cases no attempt was made to differentiate
between cytosolic and membrane bound
cellular enzyme activities and therefore
these studies can not provide direct evidence that any of the assayed enzyme activities are plasma membrane or even intracellular membrane markers.
STUDIES WITH PURIFIED MEMBRANES
Several years ago Bodnaryk et al. (1974)
presented clear histochemical evidence that
7-glutamyl transpeptidase was associated
with the brush border of Musca domestka
larval midgut and five years later Wolfersberger (1979) reported the presence of K+modulated ATPase activity in a plasma
membrane preparation from the posterior
midgut of Manduca sexta larvae. However,
it was not until the following year that two
papers were published describing attempts
to purify and enzymatically characterize
specific portions of plasma membranes
from insect midgut cells (Ferreira and
Terra, 1980; Hanozet et al., 1980). Both
of these groups used techniques which were
developed for the isolation of brush border
membranes from mammalian intestinal
cells (Schmitz et al., 1973) to produce preparations from insect larval midguts that
were enriched in alkaline phosphatase
activity (Tables 2, 3). Alkaline phosphatase
is a widely used enzyme marker for vertebrate epithelial cell microvilli membrane
(Kenny and Booth, 1978) which we have
seen (Table 1) is restricted to the cells of
R. americana larval midgut.
Ferreira and Terra (1980) assayed their
preparations for a dozen different enzyme
activities and found that cellobiase, a- and
/3-galactosidase as well as a- and /3-glucosidase, like alkaline phosphatase, were
enriched more than fourfold in the microvilli membranes relative to the homogenate. Unfortunately, they did not report
assaying their membrane preparations for
any enzymes, except acid phosphatase, that
are known to be, at least in mammalian
cells, markers for cell parts other than
plasma membrane. Because of this omission we have no way of estimating the
extent of contamination of their microvillar preparation by intracellular membranes and organelles, with the possible
TABLE 2. Specific enzyme activities in crude homogenales
and microvilli from Rhynchosciara americana larval
midguts.
Specific activity
Enzyme
Alkaline
phosphatase
Acid phosphatase
Carboxypeptidase
Cellobiase
a-Galactosidase
/3-Galactosidase
a-Glucosidase
/3-Glucosidase
Glycylglycine
dipeptidase
Leucine
aminopeptidase
Maltase
Trehalase
Homogenate
Microvilli
Enrichment
factor
37.5
230.0
6.13
13.0
349.0
57.3
1.2
232.0
83.7
58.6
114.0
4.2
469.0
399.0
6.2
1,134.0
399.0
297.0
250.0
0.32
1.34
6.96
5.17
4.89
4.77
5.07
2.19
1,583.0
4,513.0
2.85
16.3
36.7
59.7
15.7
3.66
0.43
Data from Ferreira and Terra (1980). Specific activities expressed as mUnits/mg protein.
exception of lysosomes. This is of particular concern because we have found that
the most difficult contaminant to remove
from M. sexta midgut plasma membrane
preparations is mitochondria (Harvey et al.,
19836). The approximately one-third
enrichment of acid phosphatase activity in
their membrane preparation relative to
their homogenate may indicate substantial
contamination of Ferreira and Terra's
microvillar preparation by intracellular
material (Sacchi et al., 1984).
Hanozet et al. (1980) did not report
assaying their microvillar preparations for
as many enzyme activities as Ferreira and
Terra, but they did assay their preparations for mitochondrial and cytosolic
markers (Table 3). Based on the enrichments of lactate dehydrogenase and cytochrome oxidase activities, their P. cynthia
microvillar preparations appear to be
nearly free from cytosolic and mitochondrial contamination. The reported enrichment of alkaline phosphatase activity in the
P. cynthia microvilli appears to be somewhat less than the enrichment of this
enzyme activity in R. americana microvillar
preparations but this may be deceptive
because before homogenization the contents and peritrophic membranes were
removed from the P. cynthia midguts. The
192
MICHAEL G. WOLFERSBERGER
TABLE 3. Specific enzyme activities in crude homogenates
and microvilli from Philosamia cynthia larval midguts.
Specific activity
Enzyme
Alkaline phosphatase
Cytochrome oxidase
Lactate dehydrogenase
Sucrase
Homogenate
Microvilli
0.064
4.66
0.096
0.242
0.306
0.19
0.005
0.646
Ennchmem
factor
4.78
0.04
0.05
2.67
Data from Hanozet et al. (1980). Enzyme activities
are expressed as nanomol/min/mg protein for cytochrome oxidase and micromol/min/mg protein for
the other enzymes.
modest enrichment of sucrase activity in P.
cynthia midgut brush border membranes is
consistent with the finding of Marzluf
(1969) that only about 60% of the total
sucrase activity in Drosophila melanogaster
homogenates partitions into particulate
fractions.
In more recent publications Ferreira and
Terra's group has reported preparing
microvilli from other portions of R. americana midgut (Ferreira et al., 1981; Ferreira
and Terra, 1982) as they continue their
studies aimed at understanding the details
of digestion in this insect larva. Table 4
summarizes some of their most recently
published findings. All five of the enzymes
assayed appear to be differentially distrib-
uted throughout the midgut epithelium and
in all cases show much higher specific activity in the absorptive gastric caeca and posterior ventriculus than in the secretory
anterior ventriculus. However, only three
of the enzymes showed more than a three
fold enrichment of specific activity in any
of the microvillar preparations. Two of
these three enzymes, alkaline phosphatase
and maltase, showed greatest enrichment
of specific activity in microvilli prepared
from the anterior ventriculus where their
specific activities were quite low while the
third enzyme, cellobiase, showed similar
enrichment of specific activity in microvilli
from all three regions of the midgut. It is
interesting to note (compare Table 2 with
Table 4) that as they gain experience in
preparing microvilli from R. americana larval midgut, the enrichment of several
enzyme activities in their preparations
decreases.
Giordana, Hanozet and Sacchi have
extended their studies of lepidopteran
midgut microvilli membrane enzymes in
two directions as part of their continuing
research on the mechanism of amino acid
absorption in this tissue. They have assayed
microvilli from P. cynthia larval midgut for
additional enzyme activities (Table 5) and
they have also prepared and begun to characterize enzymatically microvilli from Bom-
T A B L E 4. Specific activity of some enzymes in homogenates and microvilli prepared from different portions of R.
americana larval midgut.
Enzyme
Midgut
portion
Specific activity
Homogenate
Enrichment
factor
2,558 + 377
1.5
0.6
334 + 21
1.2
1,258 + 138
1.3
449 + 57
Carboxypeptidase A
0.7
66 + 18
1.0
181 ± 61
2.8
Cellobiase
166 ± 49
2.5
20 + 5
3.2
211+32
1.5
Maltase
45 + 6
4.4
31+5
1.4
47 + 7
3.0
Alkaline
43.9
GC
AV
4.2
3.1
phosphatase
13.1
PV
3.6
12.1
44.0
Alkaline phosphatase data from Ferreira et al. (1981), other data from Ferreira and Terra (1982). All
enzyme activities are expressed as mUnits/mg protein. Except for alkaline phosphatase, the mean ± SD (n
= 2) is given. GC, gastric caeca; AV, anterior ventriculus; PV, posterior ventriculus.
Leucine
aminopeptidase
GC
AV
PV
GC
AV
PV
GC
AV
PV
GC
AV
PV
1,703 ± 103
516 + 18
1,063 ± 46
349 ± 64
100 ± 22
179 ± 36
59 ± 9
8 ± 1
66 ± 13
31 ± 4
7+ 3
33 ± 6
14.4
Microvilli
ENZYMES OF INSECT CELL MEMBRANES
TABLE 5.
193
Enrichment of enzyine activities m microvilli membranes from Philosamia cynthia larval midguts
Specific activity
Enzyme
Maltase
Sucrase
Alkaline
phosphatase
Leucine
aminopeptidase
-y-Glutamyl
transferase
Homogenate
Microvilli
0.052 ± 0.012
0.215 ± 0.037
0.475 ± 0.051
0.045 ± 0.009
0.504 ± 0.114
4.290 ± 0.069
0.9
2.3
9.0
0.232 ± 0.011
2.232 ± 0.216
9.6
0.003 ± 0.001
0.027 ± 0.003
14.3
factor
Data from Giordana et al. (1982). Enzyme activities are expressed as micromol/min/mg protein;
mean ± SE (n = 5).
byx mori larval midgut. The preparation of
microvilli membranes from the B. mori larval midgut requires additional purification
steps in order to produce a preparation
comparable to that obtained from P. cynthia. They have assayed microvillar preparations from larvae of both species of
insects for maltase, sucrase and trehalase
but have found that only sucrase is enriched
in their membrane vesicle preparations
(Giordana et al., 1982; Sacchi et al., 1984).
They have found that leucine aminopeptidase activity is slightly more enriched than
alkaline phosphatase activity in P. cynthia
microvillar preparations and that the specific activity of -y-glutamyl transferase
(transpeptidase) is increased more than that
of any other enzyme tested (Table 5). This
latter observation is a biochemical confirmation of Bodnaryk's histochemical result
which makes this enzyme, along with alkaline phosphatase, a strong candidate for
any list of marker enzymes for insect intestinal cell brush border membranes. The
large increase in specific alkaline phosphatase activity between 1980 (Table 3) and
1982 (Table 5) is apparently due to their
having used frozen material in their earlier
studies (Giordana, personal communication).
Several years ago Dr. Moira Cioffi and
I, with encouragement and support from
Prof. W. R. Harvey, set out to isolate the
apical membrane from goblet cells of Manduca sexta larval midgut in order to study
its electrogenic cation pump (Wolfersberger et al., 1982) in cell-free systems. The
midgut of this larva, like that of other lep-
idopterans, is composed of two distinctly
different types of mature cells, columnar
cells and goblet cells, that are specialized
for different physiological functions (Cioffi,
1979, 1984). Furthermore, the cells of M.
sexta midgut, like those of other arthropod
epithelia, are joined to one another over a
substantial portion of their lateral surfaces
by septate junctions (Hakim, 1984). Using
conventional techniques, developed for
plasma membrane isolation from mammalian cells, we were able to proceed as far
as isolation of a partially purified columnar
cell apical membrane (microvilli) fraction,
a highly purified lateral membrane fraction, and a third fraction composed mainly
of both basal membranes and goblet cell
apical membranes (Harvey et al., 1981).
Numerous unsuccessful attempts to separate goblet cell apical membranes from
basal membranes forced us to reexamine
our approach and consider developing
methods specifically designed for isolation
of plasma membranes from lepidopteran
midgut epithelial cells.
Microscopic examination of pieces of
posterior midgut epithelium that had been
subjected for various times to various treatments intended to disrupt the tissue
revealed that brief sonication appeared to
disrupt only the apical brush border of the
epithelium. However, as the time intervals
to which the tissue was exposed to ultrasonic vibrations were lengthened both types
of cells were disrupted in an apparently
stepwise manner. In the course of following up on this exciting lead, we discovered
that, as predicted by Cioffi (1979), if we
194
MICHAEL G. WOLFERSBERGER
first dissected the epithelial cells from the
underlying longitudinal muscles the highly
folded epithelium became a flat sheet. This
dissection allowed much better control of
the process of cell disruption because cells
in all parts of the epithelium were similarly
exposed to disruptive forces during sonication. However, even using small, flat, and
uniformly sized pieces of tissue we were
still unable to control the sonication finely
enough to strip only one portion of the
plasma membrane from one type of epithelial cell at a time. Further experimentation revealed that we were able to
approach this degree of control if we substituted the more easily modulated sheer
forces generated by manually pipetting a
suspension of bits of epithelia for the forces
generated by the ultrasonic probe. With
methods at hand to disrupt the posterior
portion of M. sexta midgut in a stepwise
manner, in which each step yielded a tissue
extract containing plasma membrane fragments from essentially only either the apical portion of columnar cells, the apical
portion of goblet cells, or the basal portion
of both types of midgut cells, there
remained only the problem of developing
methods to separate the plasma membrane
fragments in each of these extracts from
contaminants of intracellular origin. Using
a knowledge of the composition of each
extract gained by electron microscopic
examination and experience gained from
years of subfractionation of midgut
extracts, we were soon able to develop
methods which consistently yielded highly
purified, by both positive microscopic and
negative enzymatic criteria, plasma membranes (CiofH and Wolfersberger, 1981,
1983; Harvey et al, 1983a).
results are limited to confirming that alkaline phosphatase and leucine aminopeptidase activities are highly enriched only in
the apical plasma membrane of midgut
columnar cells (microvilli) (Table 6). The
enrichment factor for neither of these
enzyme activities is exceptionally high
because our homogenates are always prepared from thoroughly washed posterior
midguts from which the peritrophic membranes and all lumenal contents have been
removed and also because the columnar
cell microvilli not only constitute a very
substantial portion (75%-80%) of the total
cell surface membrane of all posterior midgut cells but also contain a substantial portion of total tissue protein. Like our purified microvilli (Table 6), none of our other
plasma membrane fractions contain more
than 1% of the succinate dehydrogenase
activity found in homogenates of M. sexta
larval posterior midgut (Cioffi and Wolfersberger, 1983).
5'-nucleotidase is a rather well established marker enzyme for plasma membranes of mammalian cells (deDuve, 1971;
Kenny and Booth, 1978). Interestingly we
find that the specific activity of this enzyme
is not only not significantly enriched in
purified midgut columnar cell microvilli
(Table 6) but also in preliminary experiments we have found that the enrichment
factor for this enzyme activity is less than
0.25 in each of our other three plasma
membrane preparations. Although the
possibility is being investigated, it seems
unlikely (Giordana et al, 1982) that
Na + ,K + -ATPase will prove to be a marker
for the basal portion of the plasma membranes of lepidopteran midgut epithelial
cells. Despite considerable interest and
With methods available to produce effort, we have not yet been able to confirm
prediction (Harvey et al.,
highly purified preparations of specific directly either the
+
-modulated
ATPase is a
19836)
that
K
portions of M. sexta larval posterior midgut
marker
for
goblet
cell
apical
membrane
or
cell membranes, one would think that identifying enzyme markers for each isolated the prediction (Wolfersberger and Gianportion of the plasma membrane of cells giacomo, 1983) that adenyl cyclase is a
of this insect epithelium should be rela- marker for basal membranes of midgut
tively simple. However, the accomplish- epithelial cells.
ment of this theoretically straightforward
CURRENT STVTUS OF THE FIELD
objective has been delayed by severe technical limitations (Harvey etnl., 1983o). Thus
Surely the most easily substantiated conat the present time our positive enzymic clusion one can draw from the studies
195
ENZYMES OF INSECT CELL MEMBRANES
TABLE 6. Specific enzyme activities m homogenates and columnar cell apical membrane fragments(microvilli}from the
posterior midgut o/Manduca sexta larvae.
Specific activity
Enzyme
Succinate dehydrogenase
5'-NucIeotidase
Alkaline phosphatase
Leucine aminopeptidase
Homogenate
294.6
3.3
54.9
54.6
±
±
±
±
Microvilli
28.2
0.4
3.4
1.2
3.6
3.7
280.5
310.2
±
±
±
±
11.4
0.8
23.9
10.8
factor
0.01
1.12
5.11
5.68
Data from Wolfersberger and Cioffi (unpublished). Enzyme activities expressed in micromol/hr/mg protein.
Mean ± SE (n = 3).
reviewed above is that the enzymology of
insect intestinal cell plasma membranes is
one field of science in which nearly all of
the important experiments remain to be
done. However, this field has progressed
to a point at which it is now possible to do
some of these experiments and there is
hope that within the next few years many
of these experiments will be done. It has
been only a little more than two years since
the first publication of methods for preparation of purified brush border membranes from insect midgut and, although
they have been reported in abstract form,
all the details of methods for preparing
other portions of insect midgut cell membranes have just been published. There is
much interest in insect epithelial tissues.
As more investigators begin applying
sophisticated techniques of tissue fractionation to insect digestive tracts, before proceeding to measure enzyme activities, we
will begin to accumulate the data required
to establish enzyme markers for plasma
membranes of insect intestinal cells. The
establishment of such enzyme markers may
in turn facilitate studies that require the
isolation of plasma membranes from other
insect epithelia.
Currently, only -y-glutamyl transpeptidase stands unchallenged as an enzyme
whose activity is restricted to the microvilli
brush border membranes of insect midgut
columnar epithelial cells. It is perhaps not
coincidental that very few investigators
appear to have studied the distribution of
this enzyme activity in insect tissues. Leucine aminopeptidase activity appears to be
localized in the columnar cell microvilli
membrane in lepidopteran midguts but its
localization in dipteran midguts is unset-
tled. Alkaline phosphatase has been shown
to be concentrated in brush border membranes from both dipteran and lepidopteran midgut cells. However, alkaline
phosphatase as well as amino peptidase and
protease activities have been reported to
be present in salivary gland secretions from
Calliphora erythrocephala larvae (Anderson,
1982). On the basis of currently available
evidence it seems unlikely that any enzume
of carbohydrate digestion will prove to be
localized exclusively in the plasma membranes of insect intestinal cells. However,
very recent results from Terra's laboratory
(Terra and Ferreira, 1983) indicate that
this generalization may not extend to glycoprotein glycosidases.
It seems inevitable that before any reasonable consensus will be reached on which
enzymes are restricted to which membranes of which type of cells, the confusion
and disagreement about the localization of
enzyme activities in insect intestinal cells
will become much greater. Since the great
diversity among insects virtually guarantees that there will be exceptions to almost
any general statement about this class of
animals, there is probably nothing we can
do to avoid this completely. However, much
controversy can probably be avoided if
investigators carefully assay their insect cell
plasma membrane preparations for activities of not only suspected membrane
marker enzymes but also enzymes that are
known to be associated with other portions
of cells.
ACKNOWLEDGMENTS
I thank Dr. D. W. Towle for inviting me
to participate in this symposium. I gratefully acknowledge the help of Dr. J. A. T.
196
MICHAEL G. WOLFERSBERGER
Dow throughout the preparation of this
manuscript. This manuscript also benefited from discussions with Drs. M. Cioffi
and W. R. Harvey as well as reviews by
Professor Harvey and Dr. Dow. I thank Dr.
R. L. Searls for his help in preparing illustrations. I also thank J. Felder and E. Feinberg for their help in preparing the typescript. The preparation of this paper and
experimental work from this laboratory was
supported in part by Research Grant AI09503 from the National Institute of
Allergy and Infectious Diseases and by
Research Incentive Fund awards from
Temple University.
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