γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

Comp. Biochem. PhysioL Vol. 97B, No. 4, pp. 76%773, 1990 Printed in Great Britain

0305-0491/90 $3.00+ 0.00 © 1990 Pergamon Press plc

~-GLUTAMYLTRANSPEPTIDASE IN ECHINODERM EGGS A N D LARVAE: FERTILIZATION-DEPENDENT A N D DEVELOPMENTALLY-INDUCED CHANGES IN SPECIFIC ACTIVITY SUSAN J. SULAKHE,*T. H. J. GILMOUR~"and V. B. PULGA *Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0 (Tel: 306 966 6530); and ~'Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0

(Received 12 June 1990) Abstract--l. y-Glutamyltranspeptidaseis present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex. 2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min~ hr) 37% increase in the activity of v-glutamyltranspeptidase in homogenate fractions. 3. Relative to these homogenate levels, the specific activity of ~-glutamyltranspeptidase are ~ 60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs. 4. The subcellular distribution of ~-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000g particulate fractions, 22% in 40,000g soluble fractions. 5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions. 6. In homogenates of Pisaster ochraceous larvae, ~-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

INTRODUCTION

The enzyme y-glutamyltranspeptidase [(5-glutamyl)peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] catalyzes the transfer of the y-glutamyl of ~,-containing donor compounds, notably glutathione, to acceptor compounds, a diverse group of substances consisting of a variety of amino acids, glutathione itself and water (Meister and Tate, 1976; Tate and Meister, 1981; Meister, 1983; Cook and Peters, 1985a, b; Allison, 1985; Cook et al., 1987). This enzyme has been found and studied in a variety of mammalian tissues (Meister and Tate, 1976; Wapnir et al., 1982; Meister, 1983; Meister and Anderson, 1983; Furakawa et aL, 1983; Kansal et al., 1986; Sulakhe and Lautt, 1987) as well as select tissues from lower ) vertebrates from Amphibia to Reptilia (Sulakhe and Lautt, 1985, 1987; Sulakhe et al., 1988). 7-Glutamyltranspeptidase activity has also been demonstrated in salmon intestine (Bell et al., 1987), fly larvae (Bodnaryk, 1972, 1974), Trypanosoma cruzi (Repetto et al., 1987), Hydra (Meister and Tate, 1976); the mold Asperigillus oryzae (Tomita et al., 1988), yeast (Mooz and Wiglesworth, 1976; Casalone et al., 1988; Kim et al., 1988); a large number and variety of bacteria (Williams and Thorne, 1954; Talalay, 1954; Milbauer and Grossowicz, 1965; Szewczuk et al., 1970; Nakayama et al., 1984a, b; Suzuki et al., 1986, 1988; Stark et al., 1988; Angele et al., 1989) and even in plants, such as the kidney CBPB 97/4--J

bean fruit (Goore and Thompson, 1967[t, b) and the pea seedling (Kawasaki et al., 1982). Although the enzyme from these sources displays cellular, tissue, species, strain and phylogenetic differences in its specific activity its physico-chemical properties and enzymatic characteristics are remarkably similar. The apparent widespread occurrence of ~,glutamyltranspeptidase coupled with its highly conserved fundamental enzymatic characteristics have led to the proposal that this enzyme is of universal occurrence in living organisms. This, however, awaits definitive evidence that ),-glutamyltranspeptidase is present in all animal and plant groups, many of which have not thus far been examined. In the animal kingdom, it is clear that while prokaryotic and vertebrate species have received considerable attention, invertebrate species have been generally ignored. We have therefore carried out a study of ~-glutamyltranspeptidase in echinoderms, the sea urchin Lytechinus pictus and the starfish Pisaster ochraceous and report for the first time not only the presence of this enzyme in the unfertilized egg, but fertilizationdependent and developmentally-induced increases in its specific activity. MATERIALS AND METHODS

All chemicals were of analytical or reagent grade and were obtained from either Sigma Chemical Company (St Louis, MO) or Fisher Scientific Company (Edmonton, Canada).

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SUSAN J. SULAKHEet al.

Lytechinus pictus (Verrill) (L. pictus ) were obtained from Pacific Biomarine Laboratories (Los Angeles), while Pisaster ochraceous (Brandt) ( P. ochraceous ) were obtained from Seacology (North Vancouver, Canada). Fisher 344 rats were purchased from Charles Rivers (Montreal, Canada). Maintenance of various animals and the acquisition of samples Adult Lytechinuspictus (Verrill) were maintained at 15°C in aquaria containing Instant Ocean (Aquarium Systems) salt solution. Eggs and sperm were released by injection of 1 ml of a salt solution of 0.1 M acetylcholine chloride through the peristomial membranes of the adult sea urchins. The eggs were stirred in enough filtered aquarium water to produce a single layer when they were decanted into culture dishes. A dilute sperm suspension (1 ml) was added to each dish and the eggs were pipetted into microcentrifuge tubes and frozen at the following time intervals: prior to fertilization, and 5, 15, 30 and 90min, and 5, 10, 24 and 48hr post-fertilization. There were three-five individual samples per time p o i n t Pisaster ochraceous (Brandt) were maintained as above. The starfish were spawned by injection into the coelomic cavity of 1 ml of a saturated solution of 1-methyl adenine in Instant Ocean, The fertilized eggs were placed in dishes containing 100 ml of filtered aquarium water at 15°C and after 24-36 hr of development the swimming blastula stages were pipetted into newly filtered water. After 2 more days of development the larvae were ready to feed on a suspension of 5000 cells of the alga Dunaliella tertiolecta (Butcher) per ml of Instant Ocean. The algal ceils were cultured in Alga-Gro seawater medium (Carolina Biological Supply Company) supplemented with 10-6g/1 house plant food (Platabbs Corporation). The developing larvae were transferred to fresh algal suspensions every 2-3 days and frozen at the following times after fertilization: 5, 10, 16, 22 and 28 days. There were three five individual samples per time point. Male Fischer 344 rats, received at 100 g in weight, were maintained in controlled animal quarters at 21°C and 60% humidity on a 12 hr:12 hr light:dark schedule. They were kept in large'stainless steel cages, five-six/cage, fed Purina rat chow and given tap water. They were acclimatized for a 3 week period and used when their body weights approached 200 g. Rats were killed by decapitation and bled. From each of three rats, the kidneys, liver, brain and intestine were removed and placed in ice cold 0.3 M sucrose, 0.5mM dithiothreitol, 10mM Tris421, pH7.6. These organs were prepared for homogenization in the following way: kidneys and liver were trimmed of connective tissue and fat, and minced finely with scissors. The cerebral cortex was dissected carefully from the whole brain and minced finely with scissors. From the intestine, the small intestine was localized. After expelling the contents of the lumen, the entire length of the intestine was opened by longitudinal incision to expose the inner surface. Intestinal epithelial cells were obtained from this surface by scraping with a scalpel. Representative 1 g portions of either tissue minces or cells were frozen and stored at - 2 0 ° C . There were three samples of each tissue, each derived from a separate rat.

homogenization was achieved using a Polytron homogenizer equipped with a large probe, using three bursts at setting 6 at 15 sec/burst with 30sec between bursts. Homogenates were filtered through cheesecloth diluted in homogenizing buffer and assayed immediately for 7-glutamyltranspeptidase activity.

Preparation of particulate and supernatant fractions from Lytechinus pictus eggs, before, and 15 rain after fertilization Individual homogenates of unfertilized eggs and eggs 15 min post-fertilization were pooled together. Representative 100#1 aliquots were removed from each and the remaining material centrifuged in an IEC B-20A preparative centrifuge at 40,000 g at 4°C for 60 min. The clear supernatants were poured off and set aside. The yellowish pellets, containing particulate material, were suspended in one-third of the starting homogenate vol in 0.9% saline using a ground glass homogenizer. Homogenate, supernatant and particulate fractions were then assayed for 7-glutamyltranspeptidase activity at both 37 and 50°C.

Determination of 7-glutamyltranspeptidase activity 7-Glutamyltranspeptidase activities of echinoderm and rat tissue fractions were determined by our modification (Sulakhe and Lautt, 1985, 1987) of the method of Naftalm et al. (1969) using L-7-glutamyl p-nitroanilide as substrate donor and glycylglycine as acceptor. The enzyme assay was carried out at pH 8.0 at routinely 37°C; or at 50°C to assess the effect of heat on the enzyme activity. The enzymatically liberated p-nitroaniline was diazotized and the absorbance of the dye, proportional to enzyme activity, was measured at 550 nm. Enzyme activity is expressed as nmol p-nitroaniline released, mg prot- ~.min-t. Protein content of the samples was determined by the method of Lowry et al. (1951) using crystalline bovine serum albumin as standard. RESULTS

~[-Glutamyltranspeptidase activity in the unfertilized egg of Lytechinus pictus: comparison to the activities in select tissues of the Fischer 344 male rats As indicated in Table 1, ~-glutamyltranspeptidase activity is readily detectable in homogenates prepared from the unfertilized eggs of L. pictus as well as homogenates prepared from small intestinal epithelial cells, kidney, cerebral cortex and liver of the male Fischer 344 rats. With respect to the rat, a tissue-dependent variation in the specific activity of),-glutamyltranspeptidase is apparent with kidney ~ small Table 1. 7-Glutamyltranspeptidase activity in unfertilized eggs of Lytechinus pictus: comparison to the activities in select tissues of the rat Homogenate enzyme activity (nmol rag- i m i n t )

Preparation of homogenates Echinoderm homogenates. Frozen samples were thawed

(1) Lytechinus pictus eggs (2) Tissues of the male Fischer 344 rats Kidney Epithelial cells of the mucosa of the small intestine Cerebral cortex Liver

slowly on ice, transferred to 15 × 100 mm plastic tubes and homogenzied in these tubes on ice with a Polytron homogenizer equipped with a small probe, using three bursts at setting 6 at 15 sec/burst with 30 sec between bursts. Homogenates were assayed immediately for 7-glutamyltranspeptidase activity. Rat tissue homogenates. Frozen 1 g representative samples of small intestinal epithelial cells, kidney, cerebral cortex and liver tissue mince were transferred to 50 ml glass beakers and thawed in 8 ml of ice cold 0.3 M sucrose, 0.5 m M DTT; 10 mM Tris-C1, pH 7.6. Keeping the samples chilled on ice,

Homogenates were prepared from four separate samples of unfertilized eggs obtained from Lytechinus pictus as well as from 1 g representative portions of the following sets of tissues obtained from three adult male Fischer 344 rats: kidneys, cerebral cortex, epithelial cells scraped from the mucosa of the small intestine and the liver. In all cases, homogenization was achieved by Polytron homogenization, 3 × 15 sec at setting 6 using the small probe. After filtration through cheesecloth, the resultant homogenates were assayed for "e-glutamyltranspeptidase activity according to our standard procedure and conditions (Sulakhe and Lautt, 1987). Results represent mean values for three-four homogenates.

3.27 + 0.025 2626.45 + 127.89 22.54 +_0.52 5.91 + 0.67 0.42 + 0.03

769

y-Glutamyltranspeptidase intestinal epithelial cells > cerebral cortex > liver. Relative to this, the specific activity of y-glutamyltranspeptidase in homogenates of L. pictus eggs is comparable to that for homogenates from rat cerebral cortex, only 0.12% of rat kidney homogenates but 7.8-fold greater than that for homogenates from liver. y-Glutamyltranspeptidase

activity in eggs

of Lytechinus

pictus before and immediately after fertilization Figure 1 depicts the specific activity of y-glutamyltranspeptidase in homogenates of L. pictus eggs before and after fertilization, with panel A tracing the changes over 24 hr post-fertilization and panel B focusing on the changes in the first 90min post-fertilization. These results indicate that fertilization induces an early rapid increase in y -glutamyltranspeptidase activity: at 15min postfertilization enzyme activity is 37% greater than that of prefertilization levels. Furthermore, this early increase is maintained for a 6 hr period following fertilization. Finally, after this time the activity of y-glutamyltranspeptidase gradually falls: by 12 hr post-fertilization, it has attained prefertilization levels; by 24 hr post-fertilization, it has dropped to only 35% of levels expressed prefertilization. Centrifugation at 40,000 g resulted in comparable distributions of homogenate protein between supernatant and particulate fractions in both unfertilized and 15 min fertilized eggs of L. pictus: 65-70% of the protein was recovered in the supernatant fractions; 30-35% of the protein was recovered in the particulate fractions. As indicated in Fig. 2, homogenate y-glutamyltranspeptidase activity was 37% higher in eggs 15 min after fertiliz-

XGT

ACTIVITY

ation than before fertilization. Nevertheless, relative to this homogenate activity, in both cases, y-glutamyltranspeptidase activities were significantly lower in supernatant fractions: Odl-fold pre- (panel A) and 0.33-fold post- (panel B) fertilization; and significantly higher in particulate fractions: 2.7-fold pre- (panel A) and 2.75-fold post- (panel B) fertilization (numbers within the bars). The recovery of y-glutamyltranspeptidase in supernatant and particulate fractions of L. pictus eggs was identical before and after fertilization (numbers above the bars). In unfertilized eggs (panel A), 22.2% of the enzyme activity was associated with the supernatant fraction, while 77.8% of the enzyme activity was found to be particulate. After fertilization (15 min) (panel B), 21.7% of y-glutamyltranspeptidase was found in the supernatant fraction, while 78.3% was found to be particulate. It is noteworthy that the particulate enzyme displays the same degree of fertilizationdependent activation as does the enzyme in homogenates, i.e. 37%. As illustrated in Fig. 3, incubation of L. pictus fractions at 50°C as opposed to 37”C, resulted in heat-induced activation of y-glutamyltranspeptidase. While the extent of this activation varied amongst fractions, it was comparable between fractions derived from unfertilized eggs (panel A) and in eggs 15 min after fertilization (panel B). Thus, as indicated by the numbers above the bars, heatinduced activation was found to be 1.72- and 1.68-fold in homogenates; 2.6- and 3.0-fold in supernatant fractions; and, 1.97- and 1.93-fold in particulate fractions of L. picfus eggs before and after fertilization, respectively.

DURING

OF

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Fig. 1. Eggs of Lyfechinas pictus were obtained before, and at a number of time points after, fertilization (5, 15, 30 and 90 min, and 6, 12 and 24 hr). Homogenates were prepared by Polytron homogenization, 3 x 15 sec. at setting 6 using the small probe. y-Glutamyltranspeptidase activity was determined at 37°C in these fractions according to our standard procedure and conditions (Sulakhe and Lautt, 1987). The inset (B) to the full figure (A) highlights the changes over 90 min. Results represent mean values + SE of the mean for 3-5 samples/group. Numbers above the mean values reflect the activity at that time point post-fertilization relative to the activity in unfertilized eggs, expressed as a per cent.

SUSAN J. SULAKI-'IEet al.

770

DISTRIBUTION

AND RECOVERY FROM

O F "~GT A C T I V I T Y

Lytechinus

A. UNFERTILIZED EGGS

B. 15 MIN POST-FERTILIZATION (78.3%)

12

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IN F R A C T I O N S

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Fig. 2. Unfertilized, and 15min post-fertilized, eggs of Lytechinus pictus were subjected to Polytron homogenization, 3 x 15 sec at setting 6 using the small probe. Homogenates were then centrifuged at 40,000 g for 60 min to obtain supernatant and particulate fractions. Homogenate (HOM), supernatant (SUP) and particulate (PART) fractions were then assayed for 7-glutamyltranspeptidase activity at 37°C according to our standard procedure and conditions (Sulakhe and Lautt, 1987). Results for unfertilized eggs are presented in panel A and results for 15 min post-fertilized eggs are presented in panel B. Numbers within the bars represent the relative activities of 7-glutamyltranspeptidase in the various fractions with that of the homogenate being set at 1.0. Numbers in brackets above the bars represent the per cent distribution of ?-glutamyltranspeptidase between supernatant and particulate fractions.

E F F E C T OF H E A T ON

~GT A C T I V I T Y

OF

Lytechinus pictus (1.93)

14 "1 A. U N F E R T I L I Z E D EGGS

B. 15 MIN P O S T FERTILIZATION -12

12

(1,97) -10

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Fig. 3. ?-Glutamyltranspeptidase activity was determined at 50°C according to our standard procedure and conditions (Sulakhe and Lautt, 1987) in the same homogenate (HOM), supernatant (SUP) and particulate (PART) fractions of unfertilized, and 15 min post-fertilized, eggs of Lytechinus pictus eggs described in Fig. 2. Results for unfertilized eggs are presented in panel A and results of 15 min post-fertilized eggs are presented in panel B. Numbers in brackets above the bars refer to the activity at 50°C relative to that at 37°C (fold stimulation by heat treatment).

),-Glutamyltranspeptidase

7-Glutamyltranspeptidase activities during larval development of Pisaster ochraceous The activities of ?-glutamyltranspeptidase in homogenares of Pisaster ochraceous larvae at various stages of larval development is depicted in Fig. 4. The activity of ),glutamyltranspeptidase was found to be lowest at larval day 5, the earliest time point examined. Thereafter, the activity steadily increased over time. Relative to the day 5 activities, activities were found to be increased, as reflected by the number above the bars, 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28 post-fertilization, respectively.During this time, larvae were observed to grow and increase in their level of complexity but not to enter into metamorphosis.

DISCUSSION The present results indicate for the first time that the enzyme 7-glutamyltranspeptidase is present in echinoderm eggs and furthermore that it displays dramatic changes in its specific activity immediately on fertilization and during the course of larval development. The level of activity expressed is in fact substantial, being comparable to that we have observed for the enzyme from rat brain. Tissuedependent differences in the activity of ?-glutamyltranspeptidase in the adult rat reported herein are in accord with our previous results attesting to the very high activity in kidney, the very low activity in liver and the intermediate level of activity of brain (Sulakhe and Lautt, 1987).

771

The observed changes in the activity of 7-glutamyltranspeptidase in the eggs of Lytechinus pictus following fertilization, occurring as they do within 24 hr, are too rapid to be explained by a change in gene expression, especially the increase which is apparent as early as 5 min post-fertilization. While this change could be effected by a change in the sub-cellular distribution of the enzyme, our results indicate that the distribution between particulate and supernatant fractions is comparable although the specific activity of the former is much greater than that of the latter in both the unfertilized and fertilized states. Indeed, as is the case for 7-glutamyltranspeptidase from other sources (Meister and Tate, 1976; Tate and Meister, 1981; Sulakhe and Lautt, 1987) the bulk of the enzyme is particulate in the eggs of Lytechinus pictus both before and after fertilization and it is quite likely that it is plasma membrane associated. Alternatively, this fertilization-induced change could reflect a fundamental alteration in the enzyme itself. Arguing against this, however, is our observation that the enzyme responds to the effects of increasing temperature in the same way pre- and post-fertilization. We have previously reported (Sulakhe and Lautt, 1987; Sulakhe, 1987) that 7-glutamyltranspeptidase from rat liver responds to increasing temperature with an increase in its specific activity: we now expand upon this observation to include the 7-glutamyltranspeptidase from echinoderms and suggest that this may be a general property of the enzyme. The reasons for the

;~GT A C T I V I T Y DURING L A R V A L D E V E L O P M E N T OF Pisaster ochraceous

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0

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4

8

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16

20

24

28

DAYS

POST-FERTILIZATION

Fig. 4. Larvae of Pisaster ochraceous were obtained 5, 10, 16, 22 and 28 days after fertilization. Homogenates were prepared from these larvae by Polytron homogenization, 3 x 15 sec at setting 6 using the small probe, 7-Glutamyltranspeptidase activity was determined at 37°C in these fractions according to our procedure and conditions (Sulakhe and Lautt, 1987). Results represent mean values + the SE of the mean for 3-5 samples/group. Numbers above the mean reflect the activity at later stages of development relative to that at 5 days post-fertilization, expressed as a fold difference.

772

SUSANJ. SULAKI-IEet al.

somewhat greater effect in supernatant vs homogenate and particulate fractions is not presently clear. As to the identity of the determinant for the rise in y-glutamyltranspeptidase following fertilization, it is quite possible that it reflects a change in its membrane environment. In support of this contention are the numerous reports describing rapid fertilizationinduced changes in the egg plasma membrane (for reviews see Charbonneau and Grandin, 1989; Eppel, 1990; Simoncini and Moody, 1990 and references therein) as well as observations that y-glutamyltranspeptidase from other sources is sensitive to membrane perturbations (Tate and Meister, 1981; Tsuchida and Sato, 1983; Allison, 1985; Cook et al., 1987). Whether these are fluidity changes, changes in membrane potential or changes in adjacent membrane components--receptors, pumps, enzymes--remains to be determined. The rapidity of the changes in enzyme activity immediately following fertilization is particularly noteworthy. There are few observations of this nature in the literature, with the exception of our own results on the very rapid response of the enzyme in rat liver plasma membranes to thyroid hormone (Sulakhe et al., 1990), The functional significance of the fertilizationinduced increment in 7-glutamyltranspeptidase activity in the egg of Lytechinus pictus is not known. In mammalian tissues, particularly liver, increased 7-glutamyltranspeptidase activities are associated with, and in liver proposed to be markers of, the proliferative phenotype (Tate and Meister, 1981; Hanigan and Pitot, 1985; Sulakhe, 1986; Sulakhe and Lautt, 1987). Whether the rise in ~-glutamyltranspeptidase activity in the sea urchin egg on fertilization is related to the onset of proliferative activity of the zygote remains to be established. During the growth and development of Pisaster ochraceous larvae, our results indicate quite clearly that the activity of ~-glutamyltranspeptidase increases steadily over time. During this period, it has been shown that the larvae increase i n size and complexity but have not metamorphosed (Gilmour, 1988; McEdward, 1984, 1986). Thus, it would appear that ?-glutamyltranspeptidase increments follow maturation of the larvae. It is not possible to determine on the basis of these studies where the enzyme is located in the developing larvae. It is known however, that y-glutamyltranspeptidase is present in intestinal cells, not only in mammalian species (Meister and Tare, 1976; Tate and Meister, 1981; Munoz-Arrebola et al., 1989) but in non-mammalian vertebrates (Kansal and Rani, 1986; Bell et al., 1987), in the mid-gut region in the insect (Bodnaryk, 1972, 1974) and in the gastric and subhypostome regions of Hydra (Meister and Tate, 1976). Since it is established that the capture and processing of food increases in its efficiency during larval development (Strathman, 1975; Fenaux, 1982; Gilmour, 1986, 1988) and is essential for larval maturation, we propose that a likely site for yglutamyltranspeptidase in the larvae might be within the digestive system and that additionally the enzyme might participate in the uptake of digestive products such as amino acids. A role for y-glutamyltranspeptidase in amino acid uptake has been proposed by Meister and co-workers (Meister, 1973; Orlowski and

Meister, 1970; Meister and Tate, 1976; Meister and Anderson, 1983). Acknowledgements--This work was supported by an

NSERC grant awarded to S. J. Sulakhe.

REFERENCES

Allison R. D. (1985) 7-Glutamyl transpeptidase: kinetics and mechanism. Meth. Enzym. 113, 419-437. Angele C., Wellman M., Thioudellet C., Guellaen G. and Siest G. (1989) Expression of rat renal gamma-glutamyl transferase cDNA in Escherichia coli. Biochem. biophys. Res. Commun. 160, 1040-1046. Bell J. G., Buddington R. K., Walton M. J. and Cowley C. B. (1987) Studies on the putative role of ~-glutamyltranspeptidase in intestinal transport of amino acids in Atlantic salmon. J. comp. Physiol. 157B, 161-169. Bodnaryk R. P. (1972) Membrane-bound y-glutamyltranspeptidase. Evidence it is a component of the amino acid site of certain neutral amino acid transport systems. Can. J. Biochem. 50, 524-528. Bodnaryk R. P., Bronskill J. F. and Fetterly J. R. (1974) Membrane-bound v-glutamyl transpeptidase and its role in phenylalanine absorption-reabsorption in the larva of Musca domestica. J. Insect Physiol. 20, 167-181. Casalone E., Diilio C., Federici G. and Posinelli M. (1988) Glutathione and glutathione metabolizing enzymes in yeast. Antonie Van Leeuwenhoek 54, 367-378. Charbonneau M. and Grandin N. (1989) The egg of Xenopus laevis: a model system for studying cell activation. Cell Diff. Devl. 28, 71-94. Cook N. D. and Peters T. J. (1985a) Purification of 7glutamyltransferase by phenyl borate affinity chromatography. Studies on the acceptor specificityof transpeptidation by rat kidney 7-glutamyltransferase. Biochim. biophys. Acta 828, 205-212. Cook N. D. and Peters T. J. (1985b) The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney y-glutamyltransferase.Biochim. biophys. Acta 832, 142-147. Cook N. D., Upperton K. P., Challis B. C. and Peters T. J. (1987) The donor specificityand the hydrolysis reaction of ~-glutamyltransferase. Biochim. biophys. Acta 914, 240-245. Epel D. (1990) The initiation of development at fertilization. Cell Diff. Devl. 29, 1-12. Fenaux L. (1982) Nutrition of larvae. In Echinoderm Nutrition (Edited by Jangoux M.), pp. 479-489. Rotterdam, Balkema. Furukawa M., Higashi T., Tateishi N., Ochi K. and Sakamoto Y. (1983) Purification and properties of bovine liver ~-glutamyltransferase. J. Biochem. 93, 839-846. Gilmour T. J. (1986) Streamlines and particle paths in the feeding of larvae of the sea urchin Lytechinus pictus. J. exp. Mar. Biol. Ecol. 95, 27-36. Gilmour T. H. J. (1988) Particle paths and the feeding behaviour of echinoderm larvae. In Echinoderm Biology (Edited by Burke R. D., Mladenove P. V., Lambert P. and Parsley R. L.), pp. 253-257. Balkema Rotterdam. Goore M. Y. and Thompson J. F. (1967a) 7-Glutamyl transpeptidase from kidney bean fruit. I, Purification and mechanism of action. Biochim. biophys. Acta 132, 15-26. Goore M. Y. and Thompson J. F. (1967b) 7-Glutamyl transpeptidase of kidney bean fruit. II. Studies on the activating effect of sodium citrate. Biochim. biophys. Acta 132, 27-32. Hanigan M. H. and Pitot H. C. (1985) Gamma-glutamyl transpeptidase---its role in hepatocarcinogenesis. Carcinogenesis 6, 165-172.

7 -Glutamyltranspeptidase Kansal V. K. and Rani R. (1986) The activity of gammaglutamyltranspeptidase in the small intestine of some species of animals at different stages of growth. Ind. J. Physiol. Pharmac. 30, 255-258. Kawasaki Y., Ogawa T. and Sasaoka K. (1982) Occurence and some properties of a novel 7-glutamyltransferase responsible for the synthesis of ),-L-glutamyl-D-alanine in pea seedlings. Biochim. biophys. Acta 716, 194-200. Kim K. H. and Ree S. G. (1988) Subunit interaction elicited by partial inactivation with L-methione sulfoximine and ATP differently affects the biosynthesis and gammaglutamyltransferase reactions catalyzed by yeast glutamine synthase. J. biol. Chem. 262, 13,051-13,054. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Meister A. (1973) On the enzymology of amino acid transport. Science 180, 33-39. Meister A. (1983) Selective modification of glutathione metabolism. Science 220, 472~,77. Meister A. and Anderson M. E. (1983) Glutathione. A. Rev. Biochem. 52, 711-760. Meister A. and Tare S. S. (1976) Glutathione and related -glutamyl-containing compounds: biosynthesis and utilization. A. Rev. Biochem. 45, 559~04. McEdward L. R. (1984) Morphometric and metabolic analysis of the growth and form of an echinopluteus. J. exp. Mar. Biol. Ecol. 82, 259-287. McEdward L. R. (1986) Comparative morphometrics of echinoderm larvae. II. Larval size, shape, growth and scaling of feeding and metabolism. J. exp. Mar. Biol. Ecol. 96, 267-286. Milbauer R. and Grossowicz N. (1965) 7-glutamyl transfer reactions in bacteria. J. gen. Microbiol. 41, 185-194. Mooz E. D. and Wigglesworth L. (1976) Evidence of the ~,-glutamyl cycle in yeast. Biochem. biophys. Res. Commun. 68, 1066-1072. Munoz-Arrebola P., Madrid J. A., Salido G. M. and Martinez de Victoria E. (1989) Modifications of ~,glutamyl transpeptidase activity in duodenal mucosa of rats treated with different antiulcer drugs. Arch. Int. PhysioL Biochim. 97, 231-234. Nakayama R., Kumagi H. and Tochikura T. (1984a) yGlutamyltranspeptidase from bacteria. Sulfur Amino Acids 7, 427~,31. Nakayama R., Kumagi H. and Tochikura T. (1984b) Purification and properties of y-glutamyltranspeptidase from Proteus mirabilis. J. Bacteriol. 160, 341-346. Naftalin L., Sexton M., Whitaker J. F. and Tracey D. (1969) A routine procedure for estimating serum gammaglutamyltranspeptidase activity. Clin. Chim. Acta 26, 293-296. Orlowski M. and Meister A. (1970) The gamma-glutamyl cycle: a possible transport system for neutral amino acids. Proc. natn. Acad. Sci. 67, 1248-1255. Repetto Y., Letelier M. E., Aldunate J. and Morello A. (1987) The gamma-glutamyltranspeptidase of Trypanosoma cruzi. Comp. Biochem. Physiol. $7B, 73-78.

773

Simoncini L. and Moody W. J. (1990). Changes in voltagedependent currents and membrane area during maturation of starfish oocytes: species differences and similarities. Devl Biol. 138, 194-202. Stark A. A., Zeigler E. and Paganbo D. A. (1988) Glutathione mutagenesis in Salmonella typhimurium is a gamma-glutamyltranspeptidase-enhanced process involving active oxygen species. Carcinogenesis 9, 771-777. Strathmann R. R. (1975) Larval feeding in echinoderms. Am. Zool. 15, 717-730. Sulakhe S. J. (1986) The activity of 7-glutamyltranspeptidase in regenerating rat liver. FEBS Lett 204, 302-306. Sulakhe S. J. (1987) ~-Glutamyltranspeptidase in dimethylbenz (a) anthracene-induced mammary adenocarcinomas and in livers of tumor bearing rats. Int. J. Biochem. 19, 509-515. Sulakhe S. J. and Lautt W. W. (1985) The activity of hepatic ~,-glutamyltranspeptidase in various animal species. Comp. Biochem. Physiol. 82B, 263-264. Sulakhe S. J. and Lautt W. W. (1987) A characterization of ),-glutamyltranspeptidase in normal, perinatal, premalignant and malignant rat liver. Int. J. Biochem. 19, 23-32. Sulakhe S. J., Pulga V. B. and Tran S. (1988) Hepatic ctI and fl adrenergic receptors in various animal species. Molec. Cell. Biochem. 83, 81-88. Sulakhe S. J., Pulga V. B. and Tran S. (1990) Modulation of ),-glutamyltranspeptidase activity in rat liver plasma membranes by thyroid hormone. Int. J. Biochem. 22, 997-1004. Suzuki H., Kumagi H., Echigo T. and Tochikura T. (1988) Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of ~-glutamyltranspeptidase. Biochem. biophys. Res. Commun. 150, 33-38. Suzuki H., Kumagi H. and Tochikura T. (1986) 7Glutamyltranspeptidase from Escherichia coli K-12: formation and localization. J. Bacteriol. 168, 1332-1335. Szewczuk A. and Mulczy K. M. (1970) Studies on gammaglutamyl peptidase from Pseudomonas aeruginosa. Arch. Immunol. Ther. Exp. 18, 515-526. Talalay P. S. (1954) Glutathione breakdown and transpeptidase reactions in Proteus vulgaris. Nature 174, 516-517. Tate S. S. and Meister A. (1981) ~-glutamyltranspeptidase: catalytic, structural and functional aspects. Molec. Cell. Biochem. 39, 357-368. Tomita K., Ito M., Yano T., Kumagai H. and Tochikura T. (1988) ~,-Glutamyltranspeptidase activity and the properties of the extracellular glutaminase from Aspergillus oryzae. Agric. BioL Chem. 52, 1159-1163. Tsuchida T. and Sato K. (1983) Purification of detergentsolubilized form and membrane-binding domain of rat ),-glutamyltransferase by immunoattinity and hydrophobic chromatography. Biochim. biophys. Acta 756, 341-348. Wapnir R. A., Mancusi V. J. and Goldstein L. A. (1982) Comparative ontogenesis of gamma-glutamyl transpeptidase in rat tissues. Experentia 38, 647~548. Williams W. J. and Thorne C. B. (1954) Biosynthesis of 7-glutamyl peptides from glutamine by a transfer reaction. J. biol. Chem. 210, 203-217.