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The distribution of phosphomonoesterases in the digestive gut of Porcellio laevis latreille (Porcellionidae, Isopoda)
Comp. Biochem. PkysioL, 1974, Vol. 48A, pp. 37.5 to 385. Pergamm Prut. printed in Great Brirain
THE DISTRIBUTION OF PHOSPHOMONOESTERASES IN THE DIGESTIVE GUT OF PORCELLIO LAEWl3 LATREILLE (PORCELLIONIDAE, ISOPODA) M. SALEEM
and M. A. ALIKHAN
Dkpartement de Biologie, Universite Laurentienne de Sudbury, Sudbury, Ontario P3E 2C6, Canada
Abstract-1. The distribution of phosphomonoesterases was examined, by histochemical and biochemical means, in the hepatopancreas, digestive gut and the gonads of the tenth instar Pwcellio laevis. 2. The digestive gut and the hepatopancreas contained the bulk of the phosphomonoesterase activities (63.45 per cent of the total in the case of acid phosphatase, and 71 per cent of the total for alkaline phosphatase), although hepatopancreas was comparatively richer (39.05 per cent of the total) in acid phosphatase, while the digestive gut was richer (41 per cent of the total) in alkaline phosphatase. 3. More than 50 per cent of the phosphomonoesterase activity in the hepatopancreatic tissue was contained in nuclei, while mitochondria and microsomes showed 26-28 and 5 per cent activities, respectively. 4. In the digestive gut, 42-44 per cent phosphatase activity was located in the soluble fraction, 8 per cent in microsomes, 30-35 per cent in mitochondria and 25-30 per cent in nuclei. 5. Acid phosphatase activity was the highest (37.14 per cent of the total) in the hindgut, while alkaline phosphatase was most prominent (44.64 per cent of the total) in the proctodael “midgut”. 6. Both gonadal and the exoskeletal tissues appeared to be the poorest sources of phosphomonoesterases. INTRODWCTION
PHOSPHOMONO~STE~S~, a group of nonspecific phosphatases, originally discovered by Suzuki et at. (cited from Sumner & Somers, 1953) in rice bran in 1907, are thought to be involved not only in the digestion of exogenous materials but also in the autolytic degradation of tissues in such processes as developmental cycle and metamorphosis in both invertebrates and vertebrates (Roche & Latreille, 1934; Kugler & Birkner, 1948; Needham, 1954; Triantaphyllopoulos & Tuba, 1959; Gazso et al., 1961; Miiller & Tore, 1962; Miiller et al., 1963; Nath & Butler, 1971). High levels of phosphatase activity have been observed in several organs of Crustatea, including cuticle (Kugler & Birkner, 1948), gonads (de Nicola, 1949), hindgut (Holdich & Ratcliffe, 1970; Saleem & Alikhan, 1973), hepatopancreas (Saleem & Alikhan, 1973), as well as in the haemolymph (Roche & Latreille, 1934) and in the epidermally secreted moulting fluid (Needham, 1954). 375
376
M. SALEEMAND
M.A.
ALIXHAN
Previous studies in our laboratory on the nature of woodlouse phosphomonoesterases were confined to measurements of acid and alkaline phosphatases in the unfractionated combined homogenates of the digestive gut and the hepatopancreas (Saleem & Alikhan, 1973). Experiments in this paper deal with the distribution of phosphomonoesterases along the length of the hepatopancreatic and the digestive gut epitheiia, as well as with the activity of these enzymes in the various subceluiar fractions of these two tissues. MATERIALS
AND
METHODS
Enzyme source and assay Porcellio laevis used in this study were drawn from a stock colony, maintained on pieces of carrot in a glass tank at 21°C and 94-100% r.h. T o minimize individual variations as far as possible only tenth instar adults were used. The determination of the instar followed the criterion outlined by Alikhan (1972). Tissue homogenates (in double distilled water) were prepared in an all-glass homogenizer, and the differential centrifugation (on tissue homogenates in cold 0.25 M sucrose solution) was performed in an International Refrigerated (Model B20) Ultracentrifuge (International Equipment Co., Needham Hts., Mass.). The densest fraction was collected as the centrifugate at 1000 g for 5 min, and was taken to comprise of nuclei, mitochondria and partially disrupted cells (Hartenstein, 1971). The supematant was recentrifuged at 10,000 g for 10 mm to obtain a centrifugate consisting of mitochondria. The resulting supernatant was recentrifuged at 40,~O g for 30 min to procure a microsomal centrifugate and a supernatant. This later supernatant, by definition, contains the soluble fraction of the cell (Hartenstein, 1971). Protein concentration in various tissue homogenates was determined by the method of Lowry et al. (1951), while the phosphomonoesterase activities were measured by the techniques outlined by Saleem & Alikhan (1973). Histochemical localization For histochemical localization of phosphatases, isopods were dissected alive in an iced Bodenstein’s (1946) Ringer. After the removal of the unwanted adhering tissue, the digestive gut and the hepatopancreas were embedded in an O.C.T. compound “Tissue-Tek” (Ames Company, Division Miles Laboratories, Elkhart, Indiana) at -30°C. Serial sections of 10-12 p thickness were cut in a Pearse cryostat (Ames Company, Division Miles Laboratories, Etkhart, Indiana), mounted on glass slides and air dried. Acid and alkaline phosphatase activities in various sections were visualized by a slight modification of Stutt’s Naphthof AS-phosphate HNF method (Pearse, 1968). The following details are of importance: for (pH 8-Z), activator = 0.08 M alkahne phosphatase activity, buffer = 0.2 M Tris-maleate magnesium chloride; for acid phosphatase activity, buffer = 0.1 N acetate buffer (pH 4.7), activator = 0.01 M magnesium chloride. RESULTS
Distribution
of phosphatases in tissues and cells
The data on the phosphomonoesterase activity of various body tissues in the tenth instar Porcellio taevh are presented in Tables 1 and 2. As is evident from these data, both acid and alkaline phosphatases were present in each of the four types of tissues examined: hepatopancreas, digestive gut, gonads and the exoskeleton. Whether the enzyme activity in gonads and that in the exoskeleton was attributable
PHOSPHOMONOI?SrERAsES IN DIGEsTIVEGUTOF PORCELLIO
LAEVIS
377
TABLE ~-ACID PHOSPHATASE ACTIVITYOF VARIOUS BODYTISSUESIN A TENTHINSTARP. lamis LATFWLLE
Tissue Hepatopancreas Gut Gonads Exoskeleton
Total protein &g/mg wet tissue) 72+3 60+4 65+4 66f4
pm p-nitrophenol/mg wet tissue
pm p-nitrophenol/mg protein
24.8 + 1.8 15.5 -+ 1.2 13.6f 1.1 9.6 & 0.41
344*4+ 7.9 253.5 _+ 6.8 212.5 + 10.62 141.1 f 5.6
Each value is the mean of measurements on forty-five to sixty isopods + S.E. Reaction mixture: 0.5 ml 5 per cent tissue homogenate, 2 ml 0.1 M acetate buffer (pH 4*7), 2 ml distilled HaO, 0.5 ml 0.0016 M disodiump-nitrophenol phosphate, incubated for 30 min at 37°C and the amount of p-nitrophenol liberated measured at 410 nm after the addition of 10 ml 0.01 M NaOH.
to the haemolymph contained in these tissues, it could not be established. However, both the gonadal and the exoskeletal tissues, possibly the haemolymph pet- se, were found to be the poorest sources of phosphatases, both in terms of specific activity and total units. The digestive gut and the hepatopancreas, on the other hand, contained the bulk of the phosphomonoesterase activity (63.45 per cent of the total in the case of acid phosphatase, Table 1, and 71 per cent of the total for alkaline phosphatase, Table 2). In these two tissues, hepatopancreas was comparatively richer in acid phosphatase (39.05 per cent of the total, 1.6 times the proportion contained in the digestive gut, about 1.8 times that in gonads and about 2.6 times that in the exoskeleton, Table l), while the digestive gut was richer in alkaline phosphatase (41 per cent of the total, about 1.4 times the proportion contained in the hepatopancreas, three times that in the gonad and about 2.7 times that in the exoskeleton, Table 2). TABLE ~-ALKALINE PHOSPHATASE ACTIVITYOF VARIOUS BODY P. la&s LATREILLE
Tissue Hepatopancreas Gut Gonads Exoskeleton
Total protein (pg/mg wet tissue) 72+3 60f4 65+4 66+4
TISSUES IN
pm p-nitrophenollmg wet tissue 12.0 16.4 5.6 6.0
f 2.0 + 0.82 f 0.2 + 0.3
THE TENTHINSTAR
pm p-nitrophenollmg protein 166*6& 4.4 266.6 + 14.65 87.5 + 4.37 88.2 + 3.2
Each value is the mean of measurements on forty-five to sixty isopods rt S.E. Reaction mixture: 0.5 ml 5 per cent tissue homogenate, 2 ml 0.2 M Tris-maleate buffer (pH 8-l), 2 ml distilled H,O, and 0.0016 M disodium p-nitrophenol phosphate, incubated for 30 min at 37°C and the amount of p-nitrophenol released measured at 410 run after the addition of 10 ml 0.01 N NaOH.
M. SALEEM
378
AND M.A.
ALIKHAN
The data on the subcellular distribution of the enzymes (Tables 3 and 4) revealed that the major portion of the phosphomonoesterase activity in the hepatopancreatic tissue was contained in nuclei (50 per cent of the total) and in mitochondria (26-28 per cent of the total), while microsomes were the poorest source (about 5 per cent of the total). In the digestive gut tissue, on the other hand, the ~hBLE~-DISTRIBUTIONOFPHOSPHOMONOES~ASESINVhRIOuSCELLULARFRACTIONSOFTHE HEPhTOPhNC~AS INTHETENTH INSTARP. ~U~~LhT~ILLE
Phosphatase unitsjmg wet hepatopancreas Acid Nuclei Mitochondria Microsomes Soluble
175 I: 18 (50.72)* 100 + 10 (28+98)* 20 rt 7 (579)* SOY!T4 (14*49)*
Alkaline 140 75 15 50
+ 22 (solo)* + 18 (26.78)* z!I 3 (5.35)* f 4 (17&S)*
Each value is the mean of measurements for twenty-four to thirty isopods f S.E. * Percentage of total homogenate activity.
TABLE
~--DISTRIBUTION OF PHOSPHOMONOESTR~SRS IN VARIOUS CELLULAR THEDIGESTWE GUT IN THE TIZNTH INSTAR P.Zaevis LATREILLE
FRACTIONS
OF
Phosphatase units/mg wet digestive gut tissue Acid Nuclei Mitochondria Microsomes Soluble
25 35 10 5s
It 3 (20.0)* + 5 (28-o)* + 4 (8.0)* z?C 5 (44.0)*
Alkaline 30+4(250)* 30 + s (250)$ 10 & 2 (78.33)* SO& 5 (4166)*
Each value is the mean of measurements for twenty-four to thirty isopods f S.E. * Percentage of to& homogenate activity.
soluble fraction showed the highest (about 42-44 per cent of the total) phosphatase activities, and microsomes the lowest (about 8 per cent of the total). The combined phosphatase activities of nuclei and mitochondria of the digestive gut epithelium accounted for about 50 per cent of the total activity observed in the alimentary canal (Table 4). HistochemicaElocalizationof phosphomonoesterases The results on the histochemical localization of phosphatase activities in the digestive gut, hepatop~creatic and the gonadal epithelia of a tenth instar P. Eat+, are presented in Figs. 1-4.
PHOSPBOMONOWlXRASES
IN
DIGESTIVE
GUT
OF
PORCEUIO
f.REVI,S
FIG. 1. Phosphomonoesterase activity of the hepatopancreatic epithelium in a tenth instar P. h.~i~. 8, S-cell; b, B-cell. (a). Acid phosphatase (X 215). (b). Alkaline phosphatase ( x 337).
FIG. 2. Alkaline phosphatase activity of the digestive gut in a tenth instar P. Levis. (a). Foregut (x 215). (b). “Midgut” ( x 215). (c). Hindgut ( x 215).
379
380
M. SALEEM AND M. A. ALIKHAN
FIG, 3. Acid phospharase activity of the digestive gut in a tenth instar P. lmmis. (a). Foregut ( x 215). (b). ‘*Midgut” ( x 215). (c). Hindgut f x 215).
fb) FIG, 4. Phospho~~~~ster~e
activity of the gonads in atenth in star P, lam&, (a). Acid phosphatase ( x 337). (b). Alkaline phosphatase ( x 337).
P~OSFHOMONO~T~S~
IN DIGIBTIVR GUT OF PfX&EI.LR?
LAEVIS
381
The tissue sections obtained from the digestive gut and the hepatopancreas developed a dark pinkish colour (Figs. l-3), confirming the presence of high phosphatase activities in the epithelia of these two organs. The gonadal sections (Fig. 41, on the other hand, developed a purplish to weak pinkish coloration, implying a weak phosphatase activity. The phosphatase-positive granules in the hepatop~cr~~c epithelium were localized inside the large secretory cells (B-C& of Frenzef, 1894), while the small, ovoid cells (the S~~~~xe~~~, or S-cells of Frenzel, 1894) appeared relatively free of enzymes (Fig. 1). In the digestive gut epi~elium, tbe enzyme granules were concentrated in the basal margin of the cells, while the apical margins of the cells did not show any enzyme activity {Figs. 2 and 3). Whereas the phosphatase activity inside the hepatopancreatic epithelium appeared to be distributed evenly (Fig. l), it seemed to vary quite a bit in the various regions of the digestive gut (Figs. 2 and 3). In order to confirm if these differences were real, a biochemical analysis of the phosphomonoesterase activities of the various regions (i.e. foregut, proctodael “midgut” and tbe hindgut) was carried out, and the results are summarized in Table 5. TABLE 5-~OSPHOMONO~TERASE
ACTIVITY OF VARIOUS REGIONS OF THE DIGESTIVE GUT IN THE
TENTH INSTAR p. h?V&
LATRBILLR
pm p-nitrophendjmg Acid phosphatase Foregut “Midgut” Hindgut
8.0 f 0~7.5(2&57)* 9.6 rt:0~93 (34*28)* 10.4 f 1.6 {37.14)*
wet tissue
Alkaline phosphatase 12.8 k 1.2 (28*57)* 20.0 + 3,l (44+64)* 12.0 rt 1.32 (26+78)*
Each value is the mean of measurements on sixty-five to seventy-five isopods f S.E. * Percentage of the total activity in the digestive gut.
It is apparent that the alkaline phosphatase activity is si~ificantly higher in the midgut region (4464 per cent of the total) than it is in either the foregut or the hindgut (P
The si~ific~ce of phosph~monoesterases to cells and org~i~s has undergone a considerable conceptual evolution since the discovery of these enzymes in 1907. 14
382
M. SALEEM AND M. A. ALIKHAN
Initial importance was placed on their role in the bone formation (Sumner & Somers, 1953). Subsequently, through a survey of the distribution of phosphatases among vertebrate species, it was concluded that they are vital not only for the formation of bones, but also for the metabolism of carbohydrates, nucleotides and phospholipids, as well as for the process of muscular contraction (see References in Triantaphyllopoulos & Tuba, 1959). In Vertebrata, Gormi (1941) indicated that these enzymes might be localized in the striated border of the intestinal epithelium. In recent years, Hugon & Borgers (1966) supported this finding using histochemical techniques adapted to the ultrastructural level. Isolation and fractionation of microvilli revealed the enzymes to be closely associated with the membrane (Miller & Crane, 1961; Holt & Miller, 1962). Similar findings have been reported in O~y~~u~Zu~~e~(Ikeda, 3959), the steelhead trout (P&ash, 1961) and in the young chick embryo (Moog, 1944). In Isopoda, alkaline phosphatase has been found to be associated with the gonads (de Nicola, 1949), and the apical and basal cytoplasm of the juvenile hindgut epithelial cells (Holdich & Ratcliffe, 1970). In the present studies, high phosphatase activities were observed in the digestive gut and the hepatopancreatic epithelia. The acid phosphatase activity was relatively higher in the hepatopancreas, while the digestive gut epithelium appeared to be comparatively richer in alkaline phosphatase. Acid phosphatase, according to Rosenbaum & Rolon (1960), Tomlinson & Warren (1960), J ennings (1962) and Osborne & iMiller (1963), is involved not only in the initial stages of the digestion of exogenous materials but also in the autolytic degradation of tissues in such processes as developmental and the moult cycle. This implies that the hepatopancreas in Isopoda probably participates in reactions involving hydrolysis and dephosphorylation of glucose phosphate to glucose, which Renaud (1949) suggested to be a probable starting point for chitin formation. The involvement of hepatopancreas in the processes of digestion is discussed by Alikhan (1972) and Sutton (1972). Less is known about the significance of alkaline phosphatase than of acid phosphatase, although its frequent localization in absorptive epithelia in higher forms is suggestive of a role in the phosphorylation of certain compounds prior to their transport across membranes (Osborne & Miller, 1963). The pattern of alkaline phosphatase distribution observed throughout the digestive gut in the present study, considered together with the findings of Alikhan (1969,1972) on the cytological changes undergone by the epithelium during the passage of food through the isopod gut, further confirms the opinion that both “midgut” and the hindgut in Porcellio are closely involved in the digestion and absorption of food material. Holdich & Ratcliffe (1970), as well as Sutton (1972), on the other hand, insist that digestion and absorption of food in Isopoda is the function of the hepatopancreas. However, their conclusion is based mainly upon structural implications. In vertebrates, phosphomonoesterases have been generally associated with microsomes (de Duve, 1959; Triantaphyllopoulos & Tuba, 1959). In Porcellio, the
PHOSPHOMONOl?STERASES
IN DIGESTIVE GUT OF PORCELLIO
LAEVIS
383
bulk of phosphatase activity, in the hepatopancreas, was found to be confined to nuclei and mitochondria. This would imply that phosphomonoesterases in the woodlouse hepatopancreas are mainly, if not entirely, produced by nuclei and mitochondria. The site of phosphomonoesterase production in the woodlouse gut is not very clear; the bulk of acid and alkaline phosphatase activities was observed in the soluble fractions. According to Triantaphyllopoulos & Tuba (1959), it is probable that the enzymes detected in the soluble fractions may have originated in the ergastoplasm. De Duve (1959) found that 97 per cent of intestinal phosphatase activity in the rabbit and 83 per cent in the guinea pig were associated with the endoplasmic reticulum. In studies on the Wistar rat (Triantaphyllopoulas & Tuba, 1959) and on Sulvelinus fontin& (Whitmore & Goldberg, 1972) it has been suggested that the digestion products of fats, carbohydrates and proteins are absorbed through the walls of the digestive gut by means other than simple diffusion, and that this involves the participation of phosphomonoesterases. The same may hold true in the case of Porcellio digestive gut. The molecular heterogeneity of alkaline phosphatases with respect to electrophoretic mobility (see References in Whitmore & Goldberg, 1972), substrate specificity (Cox & Griffin, 1967) and inhibition by L-phenylalanine (Linscheer et al., 1971), and that of acid phosphatases with respect to pH optima (Tomlinson & Warren, 1960), and inhibition by EDTA, formaldehyde and the amino acid cysteine (Tomlinson & Warren, 1960) are well established. Prakash (1961) in his studies on the phosphomonoesterases in the steelhead trout showed that the phosphatases contained in the nuclei and mitochondria differed somewhat in their pH requirements from those located in the brush border. It is probable that in Porcellio the phosphatases procured from the digestive gut and hepatopancreatic nuclei and mitochondria are different from those localized in the microsomes of these two tissues. However, more work is needed to support this contention. Acknowledgements-The work was supported by Grant No. A3149 from the National Research Council of Canada. REFERENCES M. A. (1969) The physiology of the woodlouse, Porcellio luewis Latreille (Porcellionidae, Peracarida)-I. Studies on the gut epithelium cytology and its relation to maltase secretion. Can. J. Zool. 47, 65-75. ALIKHAN M. A. (1972) Changes in the hepatopancreas metabolic reserves of Porcellio laewis Latreille during starvation and the moult cycle. Am. Midl. Nut. 87, 503-514. BODENSTEIN D. (1946) Investigation on the locus of action of DDT in flies (Drosophila). Biol. Bull. mar. biol. Lab., Wood Hole 90, 148-157. Cox R. P. & GRIFFIN M. J. (1967) Alkaline phosphatase-I. Comparison of the physical and chemical properties of enzyme preparations from mammalian cell culture, various animal tissues, and Escherichia coli. Archs Biochem. Biophys. 122,X2-562. DE DWE C. (1959) Lysosomes, a new group of subcellular particles. In Subcellular Particles (Edited by HAYASHI T.), pp. 128-158. Ronald Press, New York. ALIKHAN
384
M. SALEEMAND M. A. ALIKHAN
FRENZEL J. (1894) Uber die Mitteldarrndriise der Crustacean. Mitt. zoiil. Stn. Neupel. 5, 50-101. GAZSO L. R., T~R~K L. J. & RAPPAY G. (1961) Contribution to the histochemistry of the nervous system of planarians. Acta Biol. Acad. Sci. Hung. 11,411-428. GORMI G. (1941) The distribution of phosphatases in normal organs and tissues. r. cell. camp. Physiol. 17, 71-83. HARTENSTEINR. (1971) Characteristics of arginase from the freshwater crayfish, Cumbarzts bartoni. Comp. Biochem. Physiol. 40B, 781-795. HOLDICH D. M. & RATCLIFFE N. A. (1970) A light and electron microscope study of the hindgut of the herbivorous isopod, Dynamene bidentata (Crustacea: Peracarida). Z. Zellforsch. mikrosk. Anal. 111,209-227. HOLT J. H. & MILLER D. (1962) The localization of phosphomonoesterase and aminopeptidases in brush border isolated from intestinal epithelial cells. Biochim. biophys. Acta 58, 239-243. HUGON J. & BORCERS M. (1966) Ultrastructural localization of alkaline phosphatase in the absorbing cells of the duodenum of mouse. J. histochem. Cytochem. 14, 629640. IKEDA A. (1959) Embryological and histochemical studies on the development of the digestive system in teleost fish, Oryzias latipes. Hiroshima J. Med. Sci. 8, 7189. JENNINGS J. B. (1962) Histochemical study of digestion and digestive enzymes in the rhynchocoelan Lineaus ruber (0. F. Miiller). Biol. Bull., mar. biol. Lab., Woods Hole 122, 63-72. KUGLER 0. E. & BIRKNERM. L. (1948) Histochemical observations of alkaline phosphatase in the integument, gastrolith sac, digestive gland and nephridium of crayfish. Physiol. ZobI. 21,105-110. LINSCHEER W. G., MALACELADAJ. R. & FISHMAN W. H. (1971) Diminished oleic acid absorption in man by L-phenylalanine inhibition of an intestinal phosphohydrolase. Nature, Lond. 231, 116-117. LOWRY 0. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MILLER D. & CRANE R. K. (1961) The digestive function of the epithelium of the small intestine--I. An intracellular locus of disaccharide and sugar phosphate ester hydrolysis. Biochim. biophys. Actu 52, 281-293. MOOG F. (1944) Localization of alkaline and acid phosphatases in the early embryogenesis of the chick. Biol. Bull., mar. biol. Lab., Woods Hole 86, 51-80. MUELLERM. & T~~Ro I. (1962) Studies on feeding and digestion in protozoa-III. Acid phosphatase activity in food vacuoles of Paramecium multimicronucleatum. J. Proto-zoiil. 9,98-102. MUELLERM., TOTH J. & T~~Ro I. (1962) Studies on feeding and digestion in protozoa-IV. Acid phosphatase and nonspecific esterase activity of food vacuoles in Amoeba proteus. Acta Biologica Acad. Sci. Hung. 13, 105-116. NATH J. & BUTLER L. (1971) Acid phosphatase during development of the black carpet beetle Attagenus megatoma (Fab.). Can.J. Biochem. 49, 311-315. NEEDHAMA. E. (1954) Properties of the minerals in the exuvia of Crustacea. Q. Jl. microsc. Sci. 95, 183-190. DE NICOLA M. (1959) Alkaline phosphatases and the cycle of nucleic acids in the gonads of some isopod crustaceans. Q.Jl. microsc. Sci. 90, 391-399. OSBORNEP. J. & MILLER A. T., JR. (1963) Acid and alkaline phosphatase changes associated with feeding, starvation and regeneration in planarians. Biol. Bull. mar. biol. Lab., Woods Hole 124, 285-292. PEARSEA. G. E. (1968) Histochemistry: Theoretical and Applied, Vol. 1. J. & A. Churchill, London.
PHOSPHOMONOESTERASES IN DIGESTIVE GUTOF PORCELLIO
LAEVIS
385
PRAKA~HA. (1960) Some observations on the distribution and pH dependence of the intestinal alkaline phosphatase of steelhead trout (S&no guirdnerii gairdnerii). Can. J. Zool. 38, 228-229. RENAUDL. (1949) Le cycle des reserves organiques chez les Crustaces Decapodes. Ann. Inst. Ocbanogr., Paris 24, 259-357. ROCHEJ. & LATREILLE M. (1934) Sur les phosphatases du sang et de l’hemolyphe. C. r. hebd. Sot. Biol. 116, 1033-1034. ROSENBAUM R. M. & ROLONC. I. (1960) Intracellular digestion and hydrolytic enzymes in the phagocytes planarians. Biol. Bull., mar. biol. Lab., Woods Hole 118, 315-323. SALEEMM. & ALIKHANM. A. (1973) Phosphomonoesterases of the digestive gut in Porcellio luewis Latreille (Porcellionidae, Isopoda). Comp. Biochem. Physiol. 45B, 91 l-921. SUMNERJ. B. & SOMERSG. F. (1953) Chemistry and Methods of Enzymes. Academic Press, New York. SUTTONS. L. (1972) Woodlice. Ginn, London. TOMLINSONN. & WARREN R. A. J. (1960) Lingcod muscle phosphomonoesterases-I. Acid phosphatases that hydrolyze p-nitrophenyl phosphate. Can. J. Biochem. 38, 605612. TRIANTAPHYLLOPOULOS E. & TUBA J. (1959) Studies on the distribution and kinetics of the alkaline phosphatase of rat small intestine. Can. J. Biochem. 37, 699-709. WHITMORED. H, & GOLDBERGE. (1972) Trout intestinal alkaline phosphatases-I. Some physical-chemical characteristics. J. exp. Zool. 37, 699-709. Key Word Index-Phosphomonoesterases; acid phosphatase; alkaline phosphatase; Porcellio laevis; Isopoda; Porcellionidae; histochemical distribution; mitochondria; gut regions; hepatopancreas; nuclei; gonads; exoskeleton.
THE DISTRIBUTION OF PHOSPHOMONOESTERASES IN THE DIGESTIVE GUT OF PORCELLIO LAEWl3 LATREILLE (PORCELLIONIDAE, ISOPODA) M. SALEEM
and M. A. ALIKHAN
Dkpartement de Biologie, Universite Laurentienne de Sudbury, Sudbury, Ontario P3E 2C6, Canada
Abstract-1. The distribution of phosphomonoesterases was examined, by histochemical and biochemical means, in the hepatopancreas, digestive gut and the gonads of the tenth instar Pwcellio laevis. 2. The digestive gut and the hepatopancreas contained the bulk of the phosphomonoesterase activities (63.45 per cent of the total in the case of acid phosphatase, and 71 per cent of the total for alkaline phosphatase), although hepatopancreas was comparatively richer (39.05 per cent of the total) in acid phosphatase, while the digestive gut was richer (41 per cent of the total) in alkaline phosphatase. 3. More than 50 per cent of the phosphomonoesterase activity in the hepatopancreatic tissue was contained in nuclei, while mitochondria and microsomes showed 26-28 and 5 per cent activities, respectively. 4. In the digestive gut, 42-44 per cent phosphatase activity was located in the soluble fraction, 8 per cent in microsomes, 30-35 per cent in mitochondria and 25-30 per cent in nuclei. 5. Acid phosphatase activity was the highest (37.14 per cent of the total) in the hindgut, while alkaline phosphatase was most prominent (44.64 per cent of the total) in the proctodael “midgut”. 6. Both gonadal and the exoskeletal tissues appeared to be the poorest sources of phosphomonoesterases. INTRODWCTION
PHOSPHOMONO~STE~S~, a group of nonspecific phosphatases, originally discovered by Suzuki et at. (cited from Sumner & Somers, 1953) in rice bran in 1907, are thought to be involved not only in the digestion of exogenous materials but also in the autolytic degradation of tissues in such processes as developmental cycle and metamorphosis in both invertebrates and vertebrates (Roche & Latreille, 1934; Kugler & Birkner, 1948; Needham, 1954; Triantaphyllopoulos & Tuba, 1959; Gazso et al., 1961; Miiller & Tore, 1962; Miiller et al., 1963; Nath & Butler, 1971). High levels of phosphatase activity have been observed in several organs of Crustatea, including cuticle (Kugler & Birkner, 1948), gonads (de Nicola, 1949), hindgut (Holdich & Ratcliffe, 1970; Saleem & Alikhan, 1973), hepatopancreas (Saleem & Alikhan, 1973), as well as in the haemolymph (Roche & Latreille, 1934) and in the epidermally secreted moulting fluid (Needham, 1954). 375
376
M. SALEEMAND
M.A.
ALIXHAN
Previous studies in our laboratory on the nature of woodlouse phosphomonoesterases were confined to measurements of acid and alkaline phosphatases in the unfractionated combined homogenates of the digestive gut and the hepatopancreas (Saleem & Alikhan, 1973). Experiments in this paper deal with the distribution of phosphomonoesterases along the length of the hepatopancreatic and the digestive gut epitheiia, as well as with the activity of these enzymes in the various subceluiar fractions of these two tissues. MATERIALS
AND
METHODS
Enzyme source and assay Porcellio laevis used in this study were drawn from a stock colony, maintained on pieces of carrot in a glass tank at 21°C and 94-100% r.h. T o minimize individual variations as far as possible only tenth instar adults were used. The determination of the instar followed the criterion outlined by Alikhan (1972). Tissue homogenates (in double distilled water) were prepared in an all-glass homogenizer, and the differential centrifugation (on tissue homogenates in cold 0.25 M sucrose solution) was performed in an International Refrigerated (Model B20) Ultracentrifuge (International Equipment Co., Needham Hts., Mass.). The densest fraction was collected as the centrifugate at 1000 g for 5 min, and was taken to comprise of nuclei, mitochondria and partially disrupted cells (Hartenstein, 1971). The supematant was recentrifuged at 10,000 g for 10 mm to obtain a centrifugate consisting of mitochondria. The resulting supernatant was recentrifuged at 40,~O g for 30 min to procure a microsomal centrifugate and a supernatant. This later supernatant, by definition, contains the soluble fraction of the cell (Hartenstein, 1971). Protein concentration in various tissue homogenates was determined by the method of Lowry et al. (1951), while the phosphomonoesterase activities were measured by the techniques outlined by Saleem & Alikhan (1973). Histochemical localization For histochemical localization of phosphatases, isopods were dissected alive in an iced Bodenstein’s (1946) Ringer. After the removal of the unwanted adhering tissue, the digestive gut and the hepatopancreas were embedded in an O.C.T. compound “Tissue-Tek” (Ames Company, Division Miles Laboratories, Elkhart, Indiana) at -30°C. Serial sections of 10-12 p thickness were cut in a Pearse cryostat (Ames Company, Division Miles Laboratories, Etkhart, Indiana), mounted on glass slides and air dried. Acid and alkaline phosphatase activities in various sections were visualized by a slight modification of Stutt’s Naphthof AS-phosphate HNF method (Pearse, 1968). The following details are of importance: for (pH 8-Z), activator = 0.08 M alkahne phosphatase activity, buffer = 0.2 M Tris-maleate magnesium chloride; for acid phosphatase activity, buffer = 0.1 N acetate buffer (pH 4.7), activator = 0.01 M magnesium chloride. RESULTS
Distribution
of phosphatases in tissues and cells
The data on the phosphomonoesterase activity of various body tissues in the tenth instar Porcellio taevh are presented in Tables 1 and 2. As is evident from these data, both acid and alkaline phosphatases were present in each of the four types of tissues examined: hepatopancreas, digestive gut, gonads and the exoskeleton. Whether the enzyme activity in gonads and that in the exoskeleton was attributable
PHOSPHOMONOI?SrERAsES IN DIGEsTIVEGUTOF PORCELLIO
LAEVIS
377
TABLE ~-ACID PHOSPHATASE ACTIVITYOF VARIOUS BODYTISSUESIN A TENTHINSTARP. lamis LATFWLLE
Tissue Hepatopancreas Gut Gonads Exoskeleton
Total protein &g/mg wet tissue) 72+3 60+4 65+4 66f4
pm p-nitrophenol/mg wet tissue
pm p-nitrophenol/mg protein
24.8 + 1.8 15.5 -+ 1.2 13.6f 1.1 9.6 & 0.41
344*4+ 7.9 253.5 _+ 6.8 212.5 + 10.62 141.1 f 5.6
Each value is the mean of measurements on forty-five to sixty isopods + S.E. Reaction mixture: 0.5 ml 5 per cent tissue homogenate, 2 ml 0.1 M acetate buffer (pH 4*7), 2 ml distilled HaO, 0.5 ml 0.0016 M disodiump-nitrophenol phosphate, incubated for 30 min at 37°C and the amount of p-nitrophenol liberated measured at 410 nm after the addition of 10 ml 0.01 M NaOH.
to the haemolymph contained in these tissues, it could not be established. However, both the gonadal and the exoskeletal tissues, possibly the haemolymph pet- se, were found to be the poorest sources of phosphatases, both in terms of specific activity and total units. The digestive gut and the hepatopancreas, on the other hand, contained the bulk of the phosphomonoesterase activity (63.45 per cent of the total in the case of acid phosphatase, Table 1, and 71 per cent of the total for alkaline phosphatase, Table 2). In these two tissues, hepatopancreas was comparatively richer in acid phosphatase (39.05 per cent of the total, 1.6 times the proportion contained in the digestive gut, about 1.8 times that in gonads and about 2.6 times that in the exoskeleton, Table l), while the digestive gut was richer in alkaline phosphatase (41 per cent of the total, about 1.4 times the proportion contained in the hepatopancreas, three times that in the gonad and about 2.7 times that in the exoskeleton, Table 2). TABLE ~-ALKALINE PHOSPHATASE ACTIVITYOF VARIOUS BODY P. la&s LATREILLE
Tissue Hepatopancreas Gut Gonads Exoskeleton
Total protein (pg/mg wet tissue) 72+3 60f4 65+4 66+4
TISSUES IN
pm p-nitrophenollmg wet tissue 12.0 16.4 5.6 6.0
f 2.0 + 0.82 f 0.2 + 0.3
THE TENTHINSTAR
pm p-nitrophenollmg protein 166*6& 4.4 266.6 + 14.65 87.5 + 4.37 88.2 + 3.2
Each value is the mean of measurements on forty-five to sixty isopods rt S.E. Reaction mixture: 0.5 ml 5 per cent tissue homogenate, 2 ml 0.2 M Tris-maleate buffer (pH 8-l), 2 ml distilled H,O, and 0.0016 M disodium p-nitrophenol phosphate, incubated for 30 min at 37°C and the amount of p-nitrophenol released measured at 410 run after the addition of 10 ml 0.01 N NaOH.
M. SALEEM
378
AND M.A.
ALIKHAN
The data on the subcellular distribution of the enzymes (Tables 3 and 4) revealed that the major portion of the phosphomonoesterase activity in the hepatopancreatic tissue was contained in nuclei (50 per cent of the total) and in mitochondria (26-28 per cent of the total), while microsomes were the poorest source (about 5 per cent of the total). In the digestive gut tissue, on the other hand, the ~hBLE~-DISTRIBUTIONOFPHOSPHOMONOES~ASESINVhRIOuSCELLULARFRACTIONSOFTHE HEPhTOPhNC~AS INTHETENTH INSTARP. ~U~~LhT~ILLE
Phosphatase unitsjmg wet hepatopancreas Acid Nuclei Mitochondria Microsomes Soluble
175 I: 18 (50.72)* 100 + 10 (28+98)* 20 rt 7 (579)* SOY!T4 (14*49)*
Alkaline 140 75 15 50
+ 22 (solo)* + 18 (26.78)* z!I 3 (5.35)* f 4 (17&S)*
Each value is the mean of measurements for twenty-four to thirty isopods f S.E. * Percentage of total homogenate activity.
TABLE
~--DISTRIBUTION OF PHOSPHOMONOESTR~SRS IN VARIOUS CELLULAR THEDIGESTWE GUT IN THE TIZNTH INSTAR P.Zaevis LATREILLE
FRACTIONS
OF
Phosphatase units/mg wet digestive gut tissue Acid Nuclei Mitochondria Microsomes Soluble
25 35 10 5s
It 3 (20.0)* + 5 (28-o)* + 4 (8.0)* z?C 5 (44.0)*
Alkaline 30+4(250)* 30 + s (250)$ 10 & 2 (78.33)* SO& 5 (4166)*
Each value is the mean of measurements for twenty-four to thirty isopods f S.E. * Percentage of to& homogenate activity.
soluble fraction showed the highest (about 42-44 per cent of the total) phosphatase activities, and microsomes the lowest (about 8 per cent of the total). The combined phosphatase activities of nuclei and mitochondria of the digestive gut epithelium accounted for about 50 per cent of the total activity observed in the alimentary canal (Table 4). HistochemicaElocalizationof phosphomonoesterases The results on the histochemical localization of phosphatase activities in the digestive gut, hepatop~creatic and the gonadal epithelia of a tenth instar P. Eat+, are presented in Figs. 1-4.
PHOSPBOMONOWlXRASES
IN
DIGESTIVE
GUT
OF
PORCEUIO
f.REVI,S
FIG. 1. Phosphomonoesterase activity of the hepatopancreatic epithelium in a tenth instar P. h.~i~. 8, S-cell; b, B-cell. (a). Acid phosphatase (X 215). (b). Alkaline phosphatase ( x 337).
FIG. 2. Alkaline phosphatase activity of the digestive gut in a tenth instar P. Levis. (a). Foregut (x 215). (b). “Midgut” ( x 215). (c). Hindgut ( x 215).
379
380
M. SALEEM AND M. A. ALIKHAN
FIG, 3. Acid phospharase activity of the digestive gut in a tenth instar P. lmmis. (a). Foregut ( x 215). (b). ‘*Midgut” ( x 215). (c). Hindgut f x 215).
fb) FIG, 4. Phospho~~~~ster~e
activity of the gonads in atenth in star P, lam&, (a). Acid phosphatase ( x 337). (b). Alkaline phosphatase ( x 337).
P~OSFHOMONO~T~S~
IN DIGIBTIVR GUT OF PfX&EI.LR?
LAEVIS
381
The tissue sections obtained from the digestive gut and the hepatopancreas developed a dark pinkish colour (Figs. l-3), confirming the presence of high phosphatase activities in the epithelia of these two organs. The gonadal sections (Fig. 41, on the other hand, developed a purplish to weak pinkish coloration, implying a weak phosphatase activity. The phosphatase-positive granules in the hepatop~cr~~c epithelium were localized inside the large secretory cells (B-C& of Frenzef, 1894), while the small, ovoid cells (the S~~~~xe~~~, or S-cells of Frenzel, 1894) appeared relatively free of enzymes (Fig. 1). In the digestive gut epi~elium, tbe enzyme granules were concentrated in the basal margin of the cells, while the apical margins of the cells did not show any enzyme activity {Figs. 2 and 3). Whereas the phosphatase activity inside the hepatopancreatic epithelium appeared to be distributed evenly (Fig. l), it seemed to vary quite a bit in the various regions of the digestive gut (Figs. 2 and 3). In order to confirm if these differences were real, a biochemical analysis of the phosphomonoesterase activities of the various regions (i.e. foregut, proctodael “midgut” and tbe hindgut) was carried out, and the results are summarized in Table 5. TABLE 5-~OSPHOMONO~TERASE
ACTIVITY OF VARIOUS REGIONS OF THE DIGESTIVE GUT IN THE
TENTH INSTAR p. h?V&
LATRBILLR
pm p-nitrophendjmg Acid phosphatase Foregut “Midgut” Hindgut
8.0 f 0~7.5(2&57)* 9.6 rt:0~93 (34*28)* 10.4 f 1.6 {37.14)*
wet tissue
Alkaline phosphatase 12.8 k 1.2 (28*57)* 20.0 + 3,l (44+64)* 12.0 rt 1.32 (26+78)*
Each value is the mean of measurements on sixty-five to seventy-five isopods f S.E. * Percentage of the total activity in the digestive gut.
It is apparent that the alkaline phosphatase activity is si~ificantly higher in the midgut region (4464 per cent of the total) than it is in either the foregut or the hindgut (P
The si~ific~ce of phosph~monoesterases to cells and org~i~s has undergone a considerable conceptual evolution since the discovery of these enzymes in 1907. 14
382
M. SALEEM AND M. A. ALIKHAN
Initial importance was placed on their role in the bone formation (Sumner & Somers, 1953). Subsequently, through a survey of the distribution of phosphatases among vertebrate species, it was concluded that they are vital not only for the formation of bones, but also for the metabolism of carbohydrates, nucleotides and phospholipids, as well as for the process of muscular contraction (see References in Triantaphyllopoulos & Tuba, 1959). In Vertebrata, Gormi (1941) indicated that these enzymes might be localized in the striated border of the intestinal epithelium. In recent years, Hugon & Borgers (1966) supported this finding using histochemical techniques adapted to the ultrastructural level. Isolation and fractionation of microvilli revealed the enzymes to be closely associated with the membrane (Miller & Crane, 1961; Holt & Miller, 1962). Similar findings have been reported in O~y~~u~Zu~~e~(Ikeda, 3959), the steelhead trout (P&ash, 1961) and in the young chick embryo (Moog, 1944). In Isopoda, alkaline phosphatase has been found to be associated with the gonads (de Nicola, 1949), and the apical and basal cytoplasm of the juvenile hindgut epithelial cells (Holdich & Ratcliffe, 1970). In the present studies, high phosphatase activities were observed in the digestive gut and the hepatopancreatic epithelia. The acid phosphatase activity was relatively higher in the hepatopancreas, while the digestive gut epithelium appeared to be comparatively richer in alkaline phosphatase. Acid phosphatase, according to Rosenbaum & Rolon (1960), Tomlinson & Warren (1960), J ennings (1962) and Osborne & iMiller (1963), is involved not only in the initial stages of the digestion of exogenous materials but also in the autolytic degradation of tissues in such processes as developmental and the moult cycle. This implies that the hepatopancreas in Isopoda probably participates in reactions involving hydrolysis and dephosphorylation of glucose phosphate to glucose, which Renaud (1949) suggested to be a probable starting point for chitin formation. The involvement of hepatopancreas in the processes of digestion is discussed by Alikhan (1972) and Sutton (1972). Less is known about the significance of alkaline phosphatase than of acid phosphatase, although its frequent localization in absorptive epithelia in higher forms is suggestive of a role in the phosphorylation of certain compounds prior to their transport across membranes (Osborne & Miller, 1963). The pattern of alkaline phosphatase distribution observed throughout the digestive gut in the present study, considered together with the findings of Alikhan (1969,1972) on the cytological changes undergone by the epithelium during the passage of food through the isopod gut, further confirms the opinion that both “midgut” and the hindgut in Porcellio are closely involved in the digestion and absorption of food material. Holdich & Ratcliffe (1970), as well as Sutton (1972), on the other hand, insist that digestion and absorption of food in Isopoda is the function of the hepatopancreas. However, their conclusion is based mainly upon structural implications. In vertebrates, phosphomonoesterases have been generally associated with microsomes (de Duve, 1959; Triantaphyllopoulos & Tuba, 1959). In Porcellio, the
PHOSPHOMONOl?STERASES
IN DIGESTIVE GUT OF PORCELLIO
LAEVIS
383
bulk of phosphatase activity, in the hepatopancreas, was found to be confined to nuclei and mitochondria. This would imply that phosphomonoesterases in the woodlouse hepatopancreas are mainly, if not entirely, produced by nuclei and mitochondria. The site of phosphomonoesterase production in the woodlouse gut is not very clear; the bulk of acid and alkaline phosphatase activities was observed in the soluble fractions. According to Triantaphyllopoulos & Tuba (1959), it is probable that the enzymes detected in the soluble fractions may have originated in the ergastoplasm. De Duve (1959) found that 97 per cent of intestinal phosphatase activity in the rabbit and 83 per cent in the guinea pig were associated with the endoplasmic reticulum. In studies on the Wistar rat (Triantaphyllopoulas & Tuba, 1959) and on Sulvelinus fontin& (Whitmore & Goldberg, 1972) it has been suggested that the digestion products of fats, carbohydrates and proteins are absorbed through the walls of the digestive gut by means other than simple diffusion, and that this involves the participation of phosphomonoesterases. The same may hold true in the case of Porcellio digestive gut. The molecular heterogeneity of alkaline phosphatases with respect to electrophoretic mobility (see References in Whitmore & Goldberg, 1972), substrate specificity (Cox & Griffin, 1967) and inhibition by L-phenylalanine (Linscheer et al., 1971), and that of acid phosphatases with respect to pH optima (Tomlinson & Warren, 1960), and inhibition by EDTA, formaldehyde and the amino acid cysteine (Tomlinson & Warren, 1960) are well established. Prakash (1961) in his studies on the phosphomonoesterases in the steelhead trout showed that the phosphatases contained in the nuclei and mitochondria differed somewhat in their pH requirements from those located in the brush border. It is probable that in Porcellio the phosphatases procured from the digestive gut and hepatopancreatic nuclei and mitochondria are different from those localized in the microsomes of these two tissues. However, more work is needed to support this contention. Acknowledgements-The work was supported by Grant No. A3149 from the National Research Council of Canada. REFERENCES M. A. (1969) The physiology of the woodlouse, Porcellio luewis Latreille (Porcellionidae, Peracarida)-I. Studies on the gut epithelium cytology and its relation to maltase secretion. Can. J. Zool. 47, 65-75. ALIKHAN M. A. (1972) Changes in the hepatopancreas metabolic reserves of Porcellio laewis Latreille during starvation and the moult cycle. Am. Midl. Nut. 87, 503-514. BODENSTEIN D. (1946) Investigation on the locus of action of DDT in flies (Drosophila). Biol. Bull. mar. biol. Lab., Wood Hole 90, 148-157. Cox R. P. & GRIFFIN M. J. (1967) Alkaline phosphatase-I. Comparison of the physical and chemical properties of enzyme preparations from mammalian cell culture, various animal tissues, and Escherichia coli. Archs Biochem. Biophys. 122,X2-562. DE DWE C. (1959) Lysosomes, a new group of subcellular particles. In Subcellular Particles (Edited by HAYASHI T.), pp. 128-158. Ronald Press, New York. ALIKHAN
384
M. SALEEMAND M. A. ALIKHAN
FRENZEL J. (1894) Uber die Mitteldarrndriise der Crustacean. Mitt. zoiil. Stn. Neupel. 5, 50-101. GAZSO L. R., T~R~K L. J. & RAPPAY G. (1961) Contribution to the histochemistry of the nervous system of planarians. Acta Biol. Acad. Sci. Hung. 11,411-428. GORMI G. (1941) The distribution of phosphatases in normal organs and tissues. r. cell. camp. Physiol. 17, 71-83. HARTENSTEINR. (1971) Characteristics of arginase from the freshwater crayfish, Cumbarzts bartoni. Comp. Biochem. Physiol. 40B, 781-795. HOLDICH D. M. & RATCLIFFE N. A. (1970) A light and electron microscope study of the hindgut of the herbivorous isopod, Dynamene bidentata (Crustacea: Peracarida). Z. Zellforsch. mikrosk. Anal. 111,209-227. HOLT J. H. & MILLER D. (1962) The localization of phosphomonoesterase and aminopeptidases in brush border isolated from intestinal epithelial cells. Biochim. biophys. Acta 58, 239-243. HUGON J. & BORCERS M. (1966) Ultrastructural localization of alkaline phosphatase in the absorbing cells of the duodenum of mouse. J. histochem. Cytochem. 14, 629640. IKEDA A. (1959) Embryological and histochemical studies on the development of the digestive system in teleost fish, Oryzias latipes. Hiroshima J. Med. Sci. 8, 7189. JENNINGS J. B. (1962) Histochemical study of digestion and digestive enzymes in the rhynchocoelan Lineaus ruber (0. F. Miiller). Biol. Bull., mar. biol. Lab., Woods Hole 122, 63-72. KUGLER 0. E. & BIRKNERM. L. (1948) Histochemical observations of alkaline phosphatase in the integument, gastrolith sac, digestive gland and nephridium of crayfish. Physiol. ZobI. 21,105-110. LINSCHEER W. G., MALACELADAJ. R. & FISHMAN W. H. (1971) Diminished oleic acid absorption in man by L-phenylalanine inhibition of an intestinal phosphohydrolase. Nature, Lond. 231, 116-117. LOWRY 0. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MILLER D. & CRANE R. K. (1961) The digestive function of the epithelium of the small intestine--I. An intracellular locus of disaccharide and sugar phosphate ester hydrolysis. Biochim. biophys. Actu 52, 281-293. MOOG F. (1944) Localization of alkaline and acid phosphatases in the early embryogenesis of the chick. Biol. Bull., mar. biol. Lab., Woods Hole 86, 51-80. MUELLERM. & T~~Ro I. (1962) Studies on feeding and digestion in protozoa-III. Acid phosphatase activity in food vacuoles of Paramecium multimicronucleatum. J. Proto-zoiil. 9,98-102. MUELLERM., TOTH J. & T~~Ro I. (1962) Studies on feeding and digestion in protozoa-IV. Acid phosphatase and nonspecific esterase activity of food vacuoles in Amoeba proteus. Acta Biologica Acad. Sci. Hung. 13, 105-116. NATH J. & BUTLER L. (1971) Acid phosphatase during development of the black carpet beetle Attagenus megatoma (Fab.). Can.J. Biochem. 49, 311-315. NEEDHAMA. E. (1954) Properties of the minerals in the exuvia of Crustacea. Q. Jl. microsc. Sci. 95, 183-190. DE NICOLA M. (1959) Alkaline phosphatases and the cycle of nucleic acids in the gonads of some isopod crustaceans. Q.Jl. microsc. Sci. 90, 391-399. OSBORNEP. J. & MILLER A. T., JR. (1963) Acid and alkaline phosphatase changes associated with feeding, starvation and regeneration in planarians. Biol. Bull. mar. biol. Lab., Woods Hole 124, 285-292. PEARSEA. G. E. (1968) Histochemistry: Theoretical and Applied, Vol. 1. J. & A. Churchill, London.
PHOSPHOMONOESTERASES IN DIGESTIVE GUTOF PORCELLIO
LAEVIS
385
PRAKA~HA. (1960) Some observations on the distribution and pH dependence of the intestinal alkaline phosphatase of steelhead trout (S&no guirdnerii gairdnerii). Can. J. Zool. 38, 228-229. RENAUDL. (1949) Le cycle des reserves organiques chez les Crustaces Decapodes. Ann. Inst. Ocbanogr., Paris 24, 259-357. ROCHEJ. & LATREILLE M. (1934) Sur les phosphatases du sang et de l’hemolyphe. C. r. hebd. Sot. Biol. 116, 1033-1034. ROSENBAUM R. M. & ROLONC. I. (1960) Intracellular digestion and hydrolytic enzymes in the phagocytes planarians. Biol. Bull., mar. biol. Lab., Woods Hole 118, 315-323. SALEEMM. & ALIKHANM. A. (1973) Phosphomonoesterases of the digestive gut in Porcellio luewis Latreille (Porcellionidae, Isopoda). Comp. Biochem. Physiol. 45B, 91 l-921. SUMNERJ. B. & SOMERSG. F. (1953) Chemistry and Methods of Enzymes. Academic Press, New York. SUTTONS. L. (1972) Woodlice. Ginn, London. TOMLINSONN. & WARREN R. A. J. (1960) Lingcod muscle phosphomonoesterases-I. Acid phosphatases that hydrolyze p-nitrophenyl phosphate. Can. J. Biochem. 38, 605612. TRIANTAPHYLLOPOULOS E. & TUBA J. (1959) Studies on the distribution and kinetics of the alkaline phosphatase of rat small intestine. Can. J. Biochem. 37, 699-709. WHITMORED. H, & GOLDBERGE. (1972) Trout intestinal alkaline phosphatases-I. Some physical-chemical characteristics. J. exp. Zool. 37, 699-709. Key Word Index-Phosphomonoesterases; acid phosphatase; alkaline phosphatase; Porcellio laevis; Isopoda; Porcellionidae; histochemical distribution; mitochondria; gut regions; hepatopancreas; nuclei; gonads; exoskeleton.