A soluble alkaline trehalase was purified from embryos and larvae of the brine shrimp, Artemia, by acetone treatment, chromatography on columns of DEAE-Sepharose Fast Flow, Con A-Sepharose and TSKgel AF-Chelate TOYOPEARL 650M, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme subjected to SDS-PAGE showed a single protein band, suggesting a molecular mass of 70,000 Da. The enzyme exhibited an apparent molecular mass of 58,000 Da on gel filtration. Endoglycosidase H digestion of the enzyme did not affect the activity of the trehalase, and resulted in a molecular mass of 66,000 Da on SDS-PAGE. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7∼4.8. The catalytic activity showed a maximum at pH 8.0, and a specific activity of 140 μmoles glucose liberated from α,α-trehalose min−1 × mg−1 was observed at 30°C. The Km value for α,α-trehalose was estimated to be 8.4 mM. Among the eleven oligosaccharides and two α-glucoside derivatives studied, the enzyme hydrolyzed only α,α-trehalose. The enzyme was maximally active at 55°C and had an activation energy of 55.8 kJ × mol−1. The enzymatic reaction was completely inhibited by 0.1 mM HgCl2. The activity of the purified enzyme was inhibited by 1 mM EDTA in the presence of 50 mM phosphate buffer, and the additions of appropriate amounts of MnCl2, MgCl2 and CaCl2 to the reaction mixture each protected the activity.
INTRODUCTION
Trehalose, a nonreducing disaccharide (α-D-glucopyranosyl (1→1)-α-D-glucopyranoside) has been found in diverse organisms including crustaceans (Elbein, 1974). Trehalose is known to be a main carbohydrate in encysted dry embryos of the brine shrimp, Artemia, which are arrested at the gastrula stage and in a state of dormancy (Dutrieu, 1960; Clegg, 1962). This major carbohydrate, which is not supplied by the parent but is synthesized by the embryo itself entering dormancy (Clegg, 1965), accounts for about 15% of the dry weight of the cysts (Clegg, 1962).
The dehydrated encysted embryos resume development upon rehydration and aerobic incubation with an adequate salinity, coinciding with a decrease in the trehalose level of the cysts accompanied by a corresponding increase in the contents of glycogen and glycerol. Thus, this disaccharide is used as a substrate of respiration, providing a large portion of the cellular energy required for the further development of the embryo (Clegg, 1964; Ewing and Clegg, 1969).
The early change in the metabolic levels mentioned above suggests a marked fluctuation of trehalase activity during the early development of Artemia. The activity of trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28), which hydrolyzes α,α-trehalose into two glucose moieties, has been observed in the embryos and larvae of Artemia. However, Boulton and Huggins (1977) found no increase of trehalase activity measured at pH 7 during Artemia development. Ballario et al. (1978) reported that encysted dry embryos of Artemia contained a trehalase that was optimally active at pH 5.6, present in insoluble form and which could be solubilized by deoxycholate treatment at high ionic strength and sonication. The reported enzyme hydrolyzed not only trehalose but also cellobiose and lactose, suggesting low specificity for trehalose or non-homogeneity. A study of a partially purified trehalase of the hydrated embryos of Artemia (Hand and Carpenter, 1986) found that this enzyme was soluble and in two active forms that interconverted when exposed to physiological transitions in pH. Vallejo (1989) showed the change of trehalase activity during the development of Artemia as well as the subcellular localization of the enzyme. The enzyme was thought to be associated with yolk granules and could be solubilized by 1% Triton X-100. The highest activity of the enzyme was detected in late nauplius.
However, most of the above authors used crude enzyme solution. Reliable methods for the detection and purification of trehalase have not yet been established, nor has anyone detected peak activity of the trehalase in the early stage of the development, although a decrease in the trehalose level of Artemia in the early stage of the development was reported by Clegg (1964), Ewing and Clegg (1969), Boulton and Huggins (1977), and Vallejo (1989).
To elucidate the dynamic activity and molecular characteristics of the trehalase in Artemia development, we purified the soluble alkaline trehalase from Artemia embryos and larvae, and investigated the trehalase activity during the development of Artemia. This report describes the purification and the properties of the trehalase from Artemia embryos and larvae as well as the developmental changes of the trehalase activity of Artemia.
MATERIALS AND METHODS
Reagents
DEAE-Sepharose Fast Flow, Con A-Sepharose, and the HiTrap™ Desalting column, Pharmalyte 3–10 and its broad pl calibration kit (pH 3–10) were purchased from Pharmacia Biotech. (Uppsala, Sweden). TSKgel AF-Chelate TOYOPEARL 650 M and the TSKgel G3000SW column were obtained from TOSOH (Tokyo, Japan). Endoglycosidase H (Streptomyces plicatus) was from Seikagaku Kogyo (Tokyo). Marker proteins for molecular weight calibration were from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and purchased from Nacalai Tesque (Kyoto, Japan).
Animals
Dehydrated cysts of Artemia were purchased from Japan Pet Drugs Co. (Tokyo, and Los Angeles, CA). Artemia cysts were from the Great Salt Lake in Utah, USA. The dry cysts (10 g) were treated with 100 ml of 7% antiformin for 1.5 hr at 4°C (Nakanishi et al., 1962). The treated cysts were washed with ice-cold distilled water, and immersed in the cold distilled water for more than 2.5 hr. The Artemia cysts thus prepared were incubated in 1,000 ml of 2% NaCl containing 0.01% each of penicillin G and streptomycin sulfate for up to 30 hr at 30°C under illumination and appropriate aeration, resulting in living nauplii of about 90%.
Enzyme assay
Trehalase activity was assayed at 30°C for 30 min by incubating an appropriate amount of enzyme in 0.5 ml of 50 mM sodium phosphate buffer (pH 7.5) and 133 mM α,α-trehalose. If the apparent optimum pH of the enzyme changed as the purification was in progress, the buffer was exchanged to 50 mM HEPES buffer (pH 7.2) or 20 mM Tricine buffer (pH 8.0) as described below. The reaction was stopped by heating the mixture in a boiling bath for 4 min. The liberated glucose was enzymatically determined as described by Dahlqvist (1964). One unit of the enzyme was defined as the amount of enzyme that produced 1 μmol of glucose per min at 30°C and optimum pH in each purification step.
Protein determination
Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard.
Electrophoresis
SDS-PAGE was carried out on a BIORAD Mini Protean II electrophoresis apparatus according to the method of Laemmli (1970) using an 8% polyacrylamide slab gel. Gels were stained for protein with Coomassie Brilliant Blue R-250. The activity of trehalase in the gel was assayed after the electrophoresis, if necessary, by incubating a non-stained sliced gel in 0.5 ml of the enzyme assay mixture as described above in the Enzyme assay section, at 37°C for 40∼60min. After the supernatant of the reaction mixture was boiled, the glucose liberated in the supernatant was enzymatically assayed by the method of Dahlqvist (1964). For the detection of the enzyme activity and preparation of the enzyme, the electrophoresis was carried out in a cold room, and the heating of the sample before electrophoresis was omitted.
Isoelectric focusing
Analytical isoelectric focusing was performed with a 5% polyacrylamide slab gel in a cold room as described by Killick (1983). The activity of trehalase in the gel was assayed after the isoelectric focusing, if necessary, as described in the Electrophoresis section above.
Estimation of molecular mass
The purified enzyme was applied to a TSKgel G3000SW column (0.75 × 60 cm) attached to a high performance liquid chromatograph, 600E (Waters, Tokyo), equilibrated with 10 mM HEPES buffer, pH 7.0, containing 0.1 M Na2SO4, 1 mM DTT and 0.0005% leupeptin. The flow rate was 1 ml/min. The positions of blue dextran and marker proteins were determined by measuring the absorption at 280 nm. The position of the trehalase was estimated by measuring the enzymatic activity at pH 8.0.
Endoglycosidase H digestion
The trehalase was treated with 3 mU of endoglycosidase H in 50mM acetate buffer, pH 5.0, containing 10 mM EDTA and 0.02% SDS for 5 hr at 37°C. The reaction mixture was lyophilized and subjected to SDS-PAGE.
Preparation of the crude enzyme
All of the operations described below were performed at 4°C unless noted.
After a 15-hr incubation, the embryos and nauplii derived from 10 g of dry cysts were collected on a nylon-mesh (40 μm), washed and homogenized in 100 ml of Buffer A (50 mM sodium phosphate buffer, pH 7.5, 1 mM EDTA, 5 mM dithiothreitol (DTT), 0.001% soybean trypsin inhibitor (STI), and 0.0005% leupeptin) by a high speed homogenizer, Physcotron (NITION, Tokyo). The supernatant with floating broken cysts resulting from centrifugation at 1,200 × g for 10min was filtered and used as a crude enzyme solution, and is hereafter referred to as the post-nuclear fraction. The solution was further centrifuged at 40,000 × g for 10 min. The supernatant and the floating orange pigment layer were pooled and homogenized, and named the post-mitochondrial fraction.
Purification of trehalase from Artemia embryos and larvae
Step 1.
Acetone treatment. A volume of acetone cooled at −20°C, equal to 8/3 of the volume of the post-mitochondrial fraction, was added to the post-mitochondrial fraction, mixed and allowed to stand in a freezer for 1 hr. The precipitate resulting from centrifugation at 1,200 × g for 10 min was washed with an appropriate volume of acetone cooled to −20°C and dried. The resulting white precipitate was suspended in 100 ml of Buffer A, sonicated for 5 min and extracted for 30 min. The extract obtained from centrifugation at 15,000 × g for 10 min was dialyzed against Solution A (1 mM EDTA, 5 mM 6-aminohexanoic acid (AHA) and 1 mM DTT) and lyophilized.
Step 2.
DEAE-Sepharose Fast Flow anion exchange column chromatography. The freeze-dried extracts obtained from two preparations were pooled and dissolved in 40 ml of 0.1 M NaCl in Buffer B (50 mM sodium phosphate buffer, pH 8.0, 1 mM EDTA, 5mM AHA, 0.001% STI, 0.0005% leupeptin and 1 mM DTT) and applied to a DEAE-Sepharose Fast Flow column (2.5 × 40.0 cm) equilibrated with 0.1 M NaCl in Buffer B. The column was washed with 320 ml of 0.1 M NaCl in Buffer B. Subsequently, the enzyme was eluted with 0.25 M NaCl in Buffer B at a flow rate of 2.7 ml/min. Fractions rich in the enzyme activity determined at pH 7.5 were pooled, added with 0.01% p-toluenesulfonyl-l-phenylalanine chloromethyl ketone (TPCK), dialyzed against Solution A, and freeze-dried.
Step 3.
Con A-Sepharose affinity chromatography. The freeze-dried material was dissolved in 7 ml of Buffer C (5 mM HEPES buffer, pH 7.2, 0.25 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 10 μM HgCl2) and applied to a Con A-Sepharose column (1.45 × 7.0 cm) equilibrated with Buffer C. The column was washed with Buffer C, and the enzyme was eluted with 0.3 M methyl α-d-mannoside in Buffer C at a flow rate of 1.4 ml/min. Fractions rich in the enzyme activity determined at pH 7.2 were pooled, added with 0.01% TPCK, dialyzed against Solution A, and lyophilized.
Step 4.
TSKgel AF-Chelate TOYOPEARL 650 M metal chelate affinity chromatography. The lyophilized dialyzate was dissolved in 6ml of distilled water, and dialyzed against Buffer D (20 mM HEPES buffer, pH 8.0, and 0.5 M NaCl) for 4 hr. The dialyzate was applied to a TSKgel AF-Chelate TOYOPEARL 650 M column (1.4 × 7 cm) pretreated with ZnCl2 and equilibrated with Buffer D. The column was washed with Buffer D, and the enzyme was eluted with 15 mM glycine in Buffer D at a flow rate of 1.7 ml/min. The active fractions determined at pH 8.0 were pooled, dialyzed against Solution A and lyophilized.
Step 5.
Preparative SDS-PAGE. The freeze-dried material was divided into 6 parts, and each part was applied to a preparative SDS-polyacrylamide mini slab gel with a thickness of 1.5 mm, respectively. After electrophoresis, a portion of the gel which corresponded to the enzyme activity determined at pH 8.0 was excised, and the enzyme was extracted in distilled water by the Physcotron homogenizer. The supernatant of the extract resulting from centrifugation at 1,200 × g for 10 min was desalted by a HiTrap™ Desalting column and lyophilized. This material was successively subjected to the second preparative SDS-PAGE.
RESULTS
The activity of the trehalase in the post-nuclear fraction from developing Artemia at different times was determined at pH 7.5, and the data obtained are shown in Fig. 1. The trehalase activity was found to be unchanged until the emergence of the stage E-1 prenauplius, followed by a 10-fold increase in its activity coinciding with the peak of the emergence of the stage E-2 prenauplius. Stage E-1 and E-2 prenauplius were defined by Nakanishi et al. (1962) (Fig. 1c). The level of the activity decreased after the hatching of the nauplius, and the activity remained at a relatively high level in the nauplii incubated for 30 hr.
Fig. 1a
The activity of the trehalase obtained from developing Artemia. Five grams of dry cysts of Artemia were incubated at 30°C in 1,000 ml of 2% NaCl-0.01% penicillin G-0.01% streptomycin sulfate, and an aliquot of 20 ml was drawn from the incubation medium at indicated times. Developing cysts in the aliquot were homogenized as described in the Materials and Methods section. The trehalase activity in the post-nuclear fraction was determined at pH 7.5. The highest activity was observed at 12 hr and was found to be 16.1 ± 0.9units/g dry cysts (arithmetic mean ± SD, n = 4). Data with longitudinal bars represent arithmetic mean ± SD (n = 3∼6, n: number of incubations).
Fig. 1b
Development of Artemia embryos and larvae. Dry cysts of Artemia (5 g) were incubated at 30°C in 1,000 ml of 2% NaCl-0.01% of penicillin G-0.01% streptomycin sulfate, and an aliquot of the incubation medium was drawn at the indicated times. The numbers of unruptured cysts, emerged stage E-1 prenauplii, emerged stage E-2 prenauplii, and hatched nauplii were counted under a stereoscopic microscope. The numbers of embryos and larvae were counted three times at each point; the total number was at least 400. Data with longitudinal bars represent arithmetic mean ± SD (n = 3∼6, n: number of incubations). •: unruptured cyst, ○: stage E-1 prenauplius, ▴: stage E-2 prenauplius, ♦: nauplius.
Fig. 1c
Developmental stages of Artemia embryo and larva. A: a cyst resuming development. B: a prenauplius at stage E-1, emerged halfway from the shell of the cyst. C: a prenauplius at stage E-2, emerged from the shell and still enclosed by an inner membrane (hatching membrane). An arrow shows cast-off outer membrane. D: a nauplius swimming freely. Scale bar = 0.1 mm.
Most of the enzyme activity was present in soluble form; the remaining part was recovered in a floating pigment layer after centrifugation. The trehalase activity in the insoluble fraction was negligible when the fraction was obtained from cysts incubated for more than 9 hr. The purification procedure and representative results are summarized in Table 1. An approximately 2,500-fold purification with a yield of 5% was obtained.
Table 1
Purification of alkaline trehalase from the developing embryos and larvae of Artemia
The purified enzyme exhibited one protein band on SDS-PAGE corresponding to a molecular mass of approximately 70,000 Da, and a peak of the activity of the purified trehalase coincided with the protein band (Fig. 2). SDS-PAGE of the enzyme under reducing and non-reducing conditions revealed the same molecular mass. The purified enzyme was applied to high performance gel filtration using a TSKgel G3000SW column. A molecular mass of 58,000 Da was calculated. These results indicate that the enzyme is a monomer lacking intramolecular disulfide bonds. After treatment of the purified enzyme with endoglycosidase H, the molecular mass of 66,000 Da was estimated on SDS-PAGE and its activity was unchanged.
Fig. 2
SDS-polyacrylamide gel electrophoresis of the Artemia trehalase. The purified Artemia trehalase weighing 3.3 μg was subjected to SDS-PAGE as described in the Materials and Methods section. Lane 1; standard proteins under non-reducing conditions. The proteins were not boiled before SDS-PAGE. a: β-galactosidase (116 kDa), b: bovine serum albumin (66 kDa), c: ovalbumin (45 kDa), d: bromphenol blue. Lane 2; purified Artemia trehalase under non-reducing conditions and without boiling before SDS-PAGE. In this case, the enzyme was active after SDS-PAGE. Lane 3; standard proteins (a,b,c) under reducing conditions; the proteins were boiled before SDS-PAGE. Lane 4; purified Artemia trehalase under reducing conditions. The enzyme was not boiled before SDS-PAGE. In this case, the enzyme was active after SDS-PAGE. Lane 5; purified Artemia trehalase under reducing conditions; the enzyme was boiled before SDS-PAGE. A lane containing 0.5 μg of the purified trehalase was subjected to SDS-PAGE under reducing conditions and without boiling; the lane was sliced, and incubated for 40 min, and the trehalase activity was determined.
The isoelectric point of the purified enzyme was determined by isoelectric focusing on a 5% polyacrylamide gel in a pH range of 3∼10 (Fig. 3). An isoelectric point of 4.7∼4.8 was estimated by measuring the enzyme activity at pH 8.0 and by staining the protein. The trehalase activity had one peak, which corresponded with one protein band.
Fig. 3
Isoelectric focusing of the Artemia trehalase. The purified trehalase from Artemia was subjected to isoelectric focusing using a 5% polyacrylamide slab gel with pH 3–10 ampholites as described in the Materials and Methods section, and stained by the method described in the calibration kit manual for pl-determinations. Lane A; 4.5 μg of purified trehalase, Lane B; pl markers, 1: lentil lectin-middle band (pl 8.45), 2: lentil lectin-acidic band (pl 8.15), 3: horse myoglobin-basic band (pl 7.35), 4: horse myoglobin-acidic band (pl 6.85), 5: human carbonic anhydrase B (pl 6.55), 6: bovine carbonic anhydrase B (pl 5.85), 7: β-lactoglobulin A (pl 5.20), 8: soybean trypsin inhibitor (pl 4.55), 9: methyl red (pl 3.75), 10: amyloglucosidase (pl 3.50). Methyl red disappeared during staining. Amyloglucosidase was scarcely stained. After isoelectric focusing, a lane containing 0.2 μg of the purified trehalase was sliced, and incubated for 60 min, and the trehalase activity was determined. Following the isoelectric focusing, four lanes without specimens were sectioned into 3 mm slices and homogenized in 2 ml of 10 mM KCl. The pH of each homogenized slice was determined at 25°C.
The dependence of the activity of the purified enzyme on the pH was investigated in a mixed buffer of 20 mM each of HEPES, MES and acetate (pH 4∼8) and in 20 mM Tricine buffer (pH 7∼9) adjusted to various pH values with NaOH. The maximum activity was observed at pH 8.0 in both buffers, and the activity measured in the latter buffer was 1.3 times higher than that in the former.
From a Lineweaver-Burk plot, 8.4 mM α,α-trehalose was calculated for the apparent Michaelis constant of the purified enzyme measured at pH 8.0. The enzyme was assayed for substrate specificity using 34.5 mM β,β-trehalose, 24 mM trehalose 6-phosphate, 20 mM each of methyl α-d-glucoside, p-nitrophenyl α-d-glucoside, sucrose, maltose, isomaltose, cellobiose, β-gentiobiose, melibiose, lactose, and raffinose. The enzyme showed a very high specificity for α,α-trehalose, and exhibited no hydrolytic action on the other substrates.
The dependence of the activity of the purified enzyme on the temperature was examined at temperatures between 25∼60°C at pH 8.0. The activity nearly doubled when the temperature rose 10°C between 25∼40°C, followed by a peak of activity at 55°C. The activation energy of the enzyme was calculated to be 55.8 kJ/mol from an Arrhenius plot of the results obtained between 25∼40°C.
Various compounds were examined for their effects on the activity of the enzyme (Table 2). Tris, sucrose, cellobiose and the aromatic derivatives of α- and β-glucosides all inhibited the activity at a concentration of 10∼20 mM. Phlorizin at 2 mM decreased the activity. The enzyme was sensitive to sulfhydryl reagents (30% and 100% inhibition by 1 mM p-hydroxymercuribenzoate (PHMB) and 0.1 mM HgCl2, respectively). Heavy metal ions such as Zn2+, Cd2+ and Cu2+ were also potent inhibitors of the trehalase. The effect of EDTA on the activity of the enzyme was examined in several conditions. EDTA at 5 mM in 50 mM HEPES buffer did not affect the activity, but it in 50 mM each of Tricine and phosphate buffer decreased the activity by 13% and 42%, respectively. The enzyme was also sensitive to 100 mM sodium phosphate buffer, resulting in a 64% inhibition of the activity. The enzyme stored in 50 mM phosphate buffer was very sensitive to EDTA, and in that condition the activity sometimes decreased to 30% by 1 mM EDTA. However, the activity was protected by the addition of appropriate amounts of Mn2+, Mg2+, and Ca2+, respectively, to the reaction mixture (Fig. 4).
Table 2
Inhibition of the trehalase activity by various compounds
Fig. 4
Inhibition of the Artemia trehalase activity by EDTA and protection by metal ions from the inhibition. Trehalase activity was assayed in 50 mM sodium phosphate buffer (pH 7.5) in the presence of 1 mM EDTA and various concentrations of metal ions. The control experiment was carried out in the absence of EDTA and metal ions, and the activity of trehalase in the control experiment was represented as 100%. ▪; with 1 mM EDTA and without metal ions, •; with 1 mM EDTA and 0.5∼5 mM MnCl2, ○; with 1 mM EDTA and 0.5∼5 mM MgCl2, 
; with 1 mM EDTA and 0.5∼5 mM CaCl2. In the case of MnCl2 and CaCl2, the precipitation of phosphate was observed during the incubation of the enzyme. The supernatant of the reaction mixture was used for the determination of the trehalase activity.
DISCUSSION
In this paper we describe a novel trehalase from Artemia embryos and larvae, which is present in soluble form and optimally active at pH 8.0, although the exact subcellular localization of the enzyme was not determined. The nature of this purified enzyme is quite different from that of the Artemia dry cyst trehalase reported earlier by Ballario et al. (1978). The latter enzyme was insoluble and active at pH 5.6. Equally important, a similarity between our Artemia alkaline trehalase and an Artemia hydrated embryo trehalase (Hand and Carpenter, 1986) should be noted. The latter authors reported that Artemia hydrated embryos contained a trehalase in soluble form which might mediate a pH-induced metabolic transition. The respiratory significance of the pH optimum of our Artemia trehalase at the resumption of Artemia development after long dormancy could be considered as follows: Artemia embryos may enter a metabolically active state through an optimization of the trehalase activity by cellular alkalinization at the resumption of development. The cellular alkalinization was previously suggested by Busa et al. (1982). We investigated this idea by elucidating the properties of the Artemia trehalase purified to homogeneity.
The coincidence of the increase in the trehalase activity with the emergence of the stage E-2 prenauplius, a phenomenon first reported here, suggests the significance of the enzyme in providing glucose to produce glycogen and glycerol, which are major respiratory substrates in the early development of Artemia (Clegg, 1964; Ewing and Clegg, 1969). This also suggests that glycerol was involved in the emergence of the prenauplius. Hygroscopic glycerol was reported to have a possible osmotic role in the rupture of the shell and the outer membrane of the Artemia cyst, i.e. the emergence (Clegg, 1964). The emerged prenauplius, still enclosed by the inner membrane (hatching membrane), protrudes from the cyst. We observed that the prenauplius began to move its antennae and mandibles within the inner membrane shortly after the emergence, piercing and tearing the inner membrane of the cyst by chance. This mechanical movement leads to the hatching of the nauplius. The moving requires much energy. Cysts incubated in distilled water (i.e., 0% NaCl) at 30°C did emerge, but the stage E-2 prenauplii did not move their antennae and mandibles, resulting in no hatching. Their trehalase activity was observed to be comparable to that of cysts ordinarily incubated in 2% NaCl (data not shown). These results indicate the significance of the enzyme in the early stage of Artemia development. Boulton and Huggins (1977) and Vallejo (1989) did not find an increase in the activity of the trehalase during the early stage of Artemia development. Differences of the extraction method of the enzyme, and of the assay method of the enzyme activity as well as of the hatchability and of the degree of synchronism in the developing Artemia from various sources might account for this discrepancy.
Considerable trehalase activity was also observed in the present study during the development of the nauplii which swam freely but did not take food. This is in parallel with the presence of a considerable amount of trehalose in nauplius (Vallejo, 1989).
Since several proteolytic activities were reported in developing Artemia (Osuna et al., 1977; Nagainis and Warner, 1979; Garesse et al., 1980; Perona and Vallejo, 1982) and neutral trehalase from yeast was described to be sensitive to proteinase (App and Holzer, 1989), many kinds of protease inhibitors were tested and introduced during the present isolation procedure. During the initial course of this work, we observed much loss of the enzyme activity when using mismatched inhibitors. The yields of the enzyme, obtained from the post-nuclear fraction through the Con A-Sepharose affinity chromatography, were improved by using the inhibitors described in Materials and Methods.
The enzyme was purified by preparative SDS-PAGE, because no inhibition of the crude enzyme activity by 0.1% SDS was observed. The activity of the purified enzyme was reduced by 50% in the presence of 0.1% SDS, and almost all of the activity was recovered after removing the SDS (data not shown).
The molecular mass of the purified trehalase from Artemia embryos and larvae was estimated to be 70 kDa by SDS-PAGE and 58 kDa by gel filtration. An earlier report (Hand and Carpenter, 1986) described the interconversion of Artemia hydrated embryo trehalase between 110 kDa at pH 8.6 and 235 kDa at pH 6.3 measured on a gel filtration column, whereas a molecular mass of 75 kDa was estimated by gel filtration for Artemia dry cyst trehalase (Ballario et al., 1978). The reported molecular mass of trehalase from different species determined by SDS-PAGE is as follows: E. coli, 58 kDa (Boos et al., 1987); yeast, 80 kDa (App and Holzer, 1989); rabbit, 66 kDa (Ruf et al., 1990); silkworm, 70 kDa (Su et al., 1993); and mealworm beetle, 62 kDa (Yaginuma et al., 1996).
The sensitivity of the purified enzyme to endoglycosidase H and the ability of the enzyme to bind to a Con A-Sepharose column suggest that the enzyme is a glycoprotein. The finding that the trehalase activity was not decreased by the endoglycosidase H treatment suggests that the carbohydrate moiety of the trehalase does not participate in its activity. The enzyme also bound a column of Lens culinalis agglutinin (LCA, Seikagaku Kogyo), and did not bind a column of wheat germ agglutinin (WGA, Seikagaku Kogyo) (data not shown). These results suggest that the sugar moiety of the trehalase is a high mannose type with fucose attached to the Asn-linked GlcNAc.
The isoelectric point of the purified enzyme was estimated to be 4.7∼4.8. This value is similar to those of trehalase from different species: silkworm, pl 4.85 (Sumida and Yamashita, 1983), rabbit intestine, pl 4.6∼4.8 (Galand, 1984), and yeast neutral trehalase, pl 4.7 (App and Holzer, 1989), but quite different from those of Artemia dry cyst trehalase (pl 6.2; Ballario et al., 1978) and of the cellular slime mold (pl 7.2∼7.3; Killick, 1983).
The calculated Km value of the purified trehalase for trehalose at pH 8.0, i.e., 8.4 mM, is smaller than those of the Artemia hydrated embryo trehalase (34.4 mM at pH 6.3 and 16.4 mM at pH 8.6; Hand and Carpenter, 1986). The Km of Artemia dry cyst trehalase was estimated to be 4.3 mM by Ballario et al. (1978). The moderate but specific affinity of our Artemia trehalase for trehalose is consistent with the high concentration of trehalose in Artemia embryos and larvae (Vallejo, 1989).
The activation energy calculated from the Arrhenius plots was 55.8 kJ/mol, which is similar to the reported values for the intestinal brush-border membrane trehalase from rabbit (46.76 kJ/mol, Galand, 1984) and for the trehalase from the cellular slime mold (50.2∼54.4 kJ/mol, Killick, 1983).
Tris, sucrose and phlorizin are known to be competitive inhibitors for trehalases from different species (rat, Nakano et al., 1977; rabbit, Galand, 1984; pig, Yoneyama, 1987) and the inhibition of the present purified Artemia trehalase by these compounds was also observed. The aromatic derivatives of α-and β-glucosides listed in Table 2 were found to be inhibitors of the Artemia trehalase. It was reported in the case of honey bee trehalase that β-glucosides were potent inhibitors of the trehalase, while α-glucosides were not (Talbot et al., 1975). The trehalase from gypsy moth was insensitive to inhibition by high concentrations of Tris, sucrose, p-nitrophenyl-β-d-glucoside or phlorizin (Valaitis and Bowers, 1993).
The SH-blocking agents 1 mM PHMB and 0.1 mM HgCl2 each inhibited the activity of the purified trehalase in the present study. These results are consistent with earlier reports of the effect of HgCl2 on the trehalase activity of different species (rat, Nakano et al., 1977; rabbit kidney, Nakano, 1982; cellular slime mold, Killick, 1983; pig, Yoneyama, 1987; yeast, App and Holzer, 1989). The activity of the purified trehalase was protected against the inhibitory effect of 0.1 mM HgCl2 by the addition of 1 mM Kl and 100 mM NaCl, respectively, to the reaction mixture (data not shown). These results are in good agreement with those reported by Nakano (1982) on kidney trehalase.
Zn2+, Cd2+ and Cu2+ at 0.1 mM concentration were potent inhibitors of the activity of the purified trehalase. The effect of EDTA on the activity of the enzyme was somewhat complicated. An inhibitory effect of EDTA on the enzyme activity was shown in Tricine and phosphate buffers, which have the ability to bind metal ions. In contrast, EDTA had no effect on the activity of the trehalase in HEPES buffer which does not bind metal ions. A high concentration (100 mM) of sodium phosphate buffer by itself inhibited the enzyme activity. These results suggest that metal ion(s) are required for the enzyme activity. The experiment designed to protect the trehalase activity against the inhibitory effect of EDTA revealed the participation of metal ions such as Mn2+, Mg2+ and Ca2+ in the activity of the purified trehalase.
Hand and Carpenter (1986) reported an inhibition of the trehalase activity of Artemia embryos by 0.1∼1.6 mM ATP. Neither Ballario et al. (1978) nor we observed such an inhibition (data not shown).
For a comparative study, cysts of Artemia from China (Japan Pet Drugs Co.) were also studied in our laboratory. The hatchability of the Chinese cysts was much lower than that of the American cysts, about 60%. The trehalase from the Chinese cysts incubated for 40 hr was purified by the same method described in Materials and Methods, and characterized. The properties of the trehalase from the Chinese cysts, i.e., the molecular mass, pH dependency, pl, etc., were much the same as those from the Great Salt Lake, USA (data not shown).
Acknowledgments
This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
