Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cellular differentiation wikipedia , lookup
Signal transduction wikipedia , lookup
Cell culture wikipedia , lookup
Cytokinesis wikipedia , lookup
Tissue engineering wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cell membrane wikipedia , lookup
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. of starving and feeding Rhynchosciara amencana larvae. Insect Biochem. 12:257-262. Giordana, B., V. F. Sacchi, and G. M. Hanozet. 1982. Intestinal amino acid absorption in lepidopteran larvae. Biochim. Biophys. Acta 692:81-88. Gooding, R. H. and B. M. Rolseth. 1976. Digestive processes of hematophagous insects. XI. Partial purification and some properties of six proteolytic enzymes from the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae). Can. J. Zool. 54:1950-1959. Hakim, R. S. and K. M. Baldwin. 1984. Cell junctions in arthropod ion-transport systems. Amer. Zool. 24:169-175. Hanozet, G. M., B. Giordana, and V. F. Sacchi. 1980. K+-dependent phenylalanine uptake in membrane vesicles isolated from the midgut of Philosatma cynthia larvae. Biochim. Biophys. Acta 596: 481-486. Hanrahan.J. 1984. Ionic permeability of insect epithelia. Amer. Zool. 24:229-240. REFERENCES Hanrahan.J. and J. Phillips. 1983. Cellular mechanisms and control of KC1 absorption in insect Anderson, O. D. 1982. Enzyme activities in the larval hindgut. J. Exp. Biol. (In press) salivary secretion of Calliphora erythrocephala. Harvey, W. R. 1982. Membrane physiology of insects. Comp. Biochem. Physiol. 72B:569-575. In R. B. Podesta (ed.), Membrane physiology ofinverBodnaryk, R. P., J. F. Bronskill, and J. R. Fetterly. tebrates, pp. 495-566. Marcel Dekker, New York. 1974. Membrane-bound 7-glutamyl transpeptidase and its role in phenylalanine absorption- Harvey, W. R., M. Cioffi, J. A. T. Dow, and M. G. Wolfersberger. 1983a. Potassium ion transport reabsorption in the larva of Musca domestica. J. ATPase in insect epithelia. J. Exp. Biol. (In press) Insect Physiol. 20:167-181. Chippendale, G. M. 1978. The functions of carbo- Harvey, W. R., M. Cioffi, and M. G. Wolfersberger. 1981. Portasomes as coupling factors in active hydrates in insect life processes. In M. Rockstein ion transport and oxidative phosphorylation. (ed.), Biochemistry of insects, pp. 1—55. Academic Amer. Zool. 21:775-791. Press, New York. Cioffi, M. 1979. The morphology and fine structure Harvey, W. R., M. Cioffi, and M. G. Wolfersberger. 19836. Chemiosmotic potassium ion pump of of the larval midgut of a moth (Manduca sexta) in insect epithelia. Am. J. Physiol. 244:R 163-R175. relation to active ion transport. Tissue and Cell House, H. L. 1974. Digestion. In M. Rockstein (ed.), 11:467-479. The physiology ofinsecta, Vol. 5, pp. 63-117. AcaCioffi, M. 1984. Comparative ultrastructure of demic Press, New York. arthropod transporting epithelia. Amer. Zool. 24: Houseman, J. G. and A. E. R. Downe. 1981. Iden139-156. tification and partial characterization of digestive Cioffi, M. and M. G. Wolfersberger. 1981. Isolation proteinases from Tnatoma phyllosoma pallidhpenof separate apical, lateral and basal membrane nis Stal (Hemiptera: Reduviidae). Comp. Biofrom cells of tobacco hornworn larval midgut. chem. Physiol. 70B:713-7l7. Amer. Zool. 21:997. (Abstr.) Kenny, A.J. and A. G. Booth. 1978. Microvilli: Their Cioffi, M. and M. G. Wolfersberger. 1983. Isolation ultrastructure, enzymology and molecular orgaof separate apical, lateral and basal plasma memnization. In P. N. Campbell and W. N. Aldridge brane from cells of an insect epithelium. A pro(eds.), Essays in biochemistry, Vol. 14, pp. 1-44. cedure based on tissue organization and ultraAcademic Press, New York. structure. Tissue and Cell 15:781-803. deDuve, C. 1971. Tissue fractionation past and pres- Law, J. H., P. E. Dunn, and K. J. Kramer. 1977. Insect proteases and peptidases. In A. Meister ent. J. Cell Biol. 50:20D-55D. (ed.), Advances in enzymology, Vol. 45, pp. 389Ferreira, C , A. F. Ribeiro, and W. R. Terra. 1981. 425. John Wiley and Sons, New York. Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications. J. Marzluf,G. A. 1969. Studies of trehalase and sucrase of Drosophila melanogaster. Arch. Biochem. BioInsect Physiol. 27:559-570. phys. 134:8-18. Ferreira, C. and W. R. Terra. 1980. Intracellular distribution of hydrolases in midgut caeca cells Ramsay, J. A. 1958. Excretion by the Malpighian tubules of the stick insect, Dixippus morosus from an insect with emphasis on plasma mem(Orthoptera, Phasmidae): Amino acids, sugars and brane-bound enzymes. Comp. Biochem. Physiol. urea. J. Exp. Biol. 35:871-891. 66B:467-473. Ferreira, C. and W. R. Terra. 1982. Function of Sacchi, V. F., G. Hanozet, and B. Giordana. 1984. a-Aminoisobutyric acid transport in the midgut midgut caeca and ventriculus: Microvilli bound of two lepidopteran larvae. J. Exp. Biol. (In press) enzymes from cells of different midgut regions ENZYMES OF INSECT CELL MEMBRANES 197 Schmitz, J., H. Preiser, D. Maestracci, B. K. Ghosh, Wigglesworth, V. B. 1972. The principles of insect physJ.J. Cerda, and R. K. Crane. 1973. Purification iology. Chapman and Hall, London. of the human intestinal brush border membrane. Wolfersberger, M. G. 1979. A potassium-modulated Biochim. Biophys. Acta 323:98-112. plasma membrane adenosine triphosphatase from Terra, W. R. and C. Ferreira. 1981. The physiologthe midgut of Manduca sexta larvae. Fed. Proc. ical role of the peritrophic membrane and tre38:242. halase: Digestive enzymes in the midgut and Wolfersberger, M. G. and K. M. Giangiacomo. 1983. excreta of starved larvae of Rhyn.chosa.ara. J. Insect Active potassium transport by isolated lepidopPhysiol. 27:325-331. teran midgut: Stimulation of net potassium flux Terra, W. R. and C. Ferreira. 1983. Further eviand elimination of the slower phase decline of dence that enzymes involved in the final stages the short circuit current. J. Exp. Biol. 102:199of digestion by Rhynchosaara do not enter the 210. endoperitrophic space. Insect Biochem. 13:143- Wolfersberger, M. G., W. R. Harvey, and M. Cioffi. 150. 1982. Transepithelial potassium ion transport in Terra, W. R., C. Ferreira, and A. G. deBianchi. 1979. insect midgut by an electrogenic alkali metal ion Distribution of digestive enzymes among the endopump. In F. Bronner, A. Kleinzeller, and C. Slayand ectoperitrophic spaces and midgut cells of man (eds.), Current topics in membranes and transRhynchosaara and its physiological significance. J. port, Vol. 16, pp. 109-133. Academic Press, New Insect Physiol. 25:487-494. York. Towle, D. 1984. Membrane-bound ATPases in Wyatt, G. R. 1967. The biochemistry of sugars and polysaccharides in insects. In J. W. L. Beament, arthropod ion-transporting tissues. Amer. Zool. J. E. Treherne, and V. B. Wigglesworth (eds.), 24:177-185. Advances in insect physiology, Vol. 4, pp. 287-360. Treherne.J. E. 1958. The digestion and absorption Academic Press, New York. of tripalmitin in the cockroach, Penplaneta americana L. J. Exp. Biol. 35:862-869.