THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 264, No. 27, Issue of September 25, pp. 16165-16169,1989 Printed in U. S. A.

Purification and Characterization of Trehalase Inhibitor from Hemolymph of the American Cockroach, Periplaneta arnericana"

(Received for publication, January 26, 1989)

Yoichi HayakawaS, Ashok P. Jahagirdars, Makoto Yaguchill, and Roger G . H. Downers From the $Biological Laboratory, The Institute of Low Temperature Science, Hokkaido University, Sapporo 060, Japan, the $Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada, and the TDiuision of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, KIA OR6. Canada

An endogenous proteinaceous inhibitor of trehalase (a,&-trehalose-1-glucohydrolase: EC 3.2.1.28) has been isolated and purified from the serum of resting adult American cockroaches, Periplaneta americana. Purification procedures involved decreasing ionic strength, gel filtration, and reversed phase high per- formance liquid chromatography. Homogeneity was confirmed by polyaclrylamide gel electrophoresis and end group analysis. The purified protein inhibited tre- halase activity in a dose-dependent manner and was estimated to have a molecular weight of 86,000 and to contain sugar chains. An automated gas-phase sequen- cer was used to determine the following sequence for the N-terminal amino acid residues: H-Ala-Ilu-Pro- Thr-Pro-His-Val-Tyr-Lys-Val-X-Val-Pro-Asp-Gly- Ala-Leu-Asn-Asp.

The hemolymph of most insects contains high concentra- tions of the nonreducing disaccharide, trehalose together with the a-glucosidase, trehalase (ap-trehalose 1-glucohydrolase: EC 3.2.1.28) (1). Seve:ral theories have been advanced to account for the anomolous co-existence of the enzyme and its substrate in insect hemolymph. These include spatial sepa- ration of the enzyme from the substrate through compart- mentalization of trehal.ase in hemocytes (2) , although the detection of trehalase activity in the serum component of whole hemolymph (3, 4) suggests that, at best, compartmen- talization provides on1.y a partial explanation for the co- existence. The pH of cockroach hemolymph drops from pH 7.5 to 6.5 during exercise, and, as this results in a 20-fold increase in trehalase activity ( 5 ) , pH-mediated activation of trehalase must be considered as an important facilitator of the trehalose/trehalase system (6, 7). The presence of a heat- labile, nondialyzable inhibitor of trehalase which functions in association with a bivadent cation was suggested in hemo- lymph of the blowfly Phormia regina (S), but the observations were not substantiated in the cockroach (3). However, evi- dence in support of the presence of a trehalase inhibitor in cockroach hemolymph is provided by the demonstration that a partially purified inactive form of hemolymph trehalase is activated both by trypsinization and by increasing the ionic strength of the assay mixture (9). The present study extends these observations through the isolation, purification, and

* This work was supported by operating and strategic grants from the Natural Sciences and Engineering Research Council of Canada and a Fellowship from the Japan Society for Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

partial characterization of an inhibitor of trehalase from serum of the American cockroach, Periplaneta americana.

MATERIALS AND METHODS

Animals-Insects were taken from a colony of the American cock- roach, P. americana, maintained in this laboratory under standard conditions (9).

Chemicals-Taurine, 0-dianisidine, glucose oxidase, peroxidase, hexokinase, and glucose-6-phosphate dehydrogenase were purchased from Sigma.

Preparation of Serum-Cockroaches were isolated in individual Petri dishes with water for 15-16 h before being injected with 5 p1 of taurine solution (16 nmol of taurine in physiological saline: 140 mM NaC1, 9.4 mM KCl, 3.0 mM CaC12, 3.6 mM NaHC03, 1.6 mM KHzPO,, 1.6 mM NaZHP04, 4.0 mM MgCl,, pH 7.0); the isolation suppressed excitation-induced activation of serum trehalase activity (8). Serum from taurine-injected cockroaches was used as a source of the treha- lase inhibitor, because injection of cockroaches with taurine reduces serum trehalase activity (9). Injected insects were held in individual Petri dishes for a further 2 h prior to hemolymph collection. The hemolymph was collected into a chilled polypropylene test tube through the wound caused by severing the base of a metathoracic appendage following injection of 100 p1 of EDTA solution (150 mM KCI, 10 mM EDTA, 6 mM l-phenyl-2-thiourea, 50 mM phosphate buffer, pH 6.8). Collected hemolymph was immediately centrifuged at 4 "C for 5 min at 500 X g and the supernatant used as the serum sample.

Isolation of Inhibition Factor-Four milliliters of serum, collected from about 60 animals, was placed in a dialyzing tube (Spectrum Medical Industries; compounds of molecular mass greater than 18,000 Da are not dialyzable) and dialyzed against 4 1' of chilled distilled water for 15-20 h. Following centrifugation at 20,000 X g for 5 min, the supernatant of the dialysate was concentrated by lyophilization.

Preparation of Trehalase from Hemocytes-Previous studies indi- cate that the same enzyme is responsible for the trehalase activity associated with serum and hemocytes (5). The trehalase used in the present study was purified from the hemocytes that precipitated during the centrifugation step of serum preparation (see above). The hemocyte pellet was sonicated for 1 min in the original volume of 50 mM phosphate buffer (pH 6.0) using a Branson Sonifier Cell Disrup- tor 200 at 45 watts. The sonicated preparation was centrifuged at 200,000 X g for 15 min at 4 "C and the supernatant applied to a DEAE-cellulose column (18 X 2.0 cm) equilibrated with 50 mM phosphate buffer (pH 7.0). The column was washed with 50 ml of buffer, and proteins were then eluted using a linear gradient of 40 ml of NaCl (0-0.5 M) in buffer. Trehalase activity was detected in the fraction eluted with 0.15 M NaCl with about 40% recovery. The eluted active fraction was then applied to a Superose 6 gel filtration column on a fast protein liquid chromatography system (Pharmacia LKB Biotechnology Inc.) and eluted with 50 mM phosphate buffer (pH 6.6) containing 0.15 M NaCl with about 38% recovery. The active fraction was used as the trehalase sample for subsequent experiments. Tre- halase was also purified from serum using the same procedures as described above. The serum trehalase was identical to the hemocyte enzyme with regard to inhibition sensitivity. Trypsinization of puri- fied enzyme and freshly collected serum was achieved according to

' The abbreviations used are: 1, liter; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

16165

16166 Purification of Trehalase Inhibitor from Insect Hemolymph the procedure described previously (9).

Enzyme Assay-Fifty microliters of the trehalase preparation (63 pg protein/ml) was incubated with 350 p1 of inhibitor solution and 150 pl of 80 mM phosphate buffer (47 mM NaC1,3.1 mM KC1, 1.0 mM CaC12, 1.2 mM NaHC03, 0.53 mM KH2P04, 0.53 mM Na,HPO,, 1.3 mM MgCI,, pH 6.6) for 3 min at 30 "C. The reaction was started by adding 50 pl of 0.66 M trehalose solution, and after a 1-h incubation at 30 'C, terminated by boiling for 3 min. Two analytical procedures were used to determine the glucose content of the reaction mixture. These were a modification of the glucose oxidase method (10) (glucose oxidase, 29,750 units/l; peroxidase 1,500 units/l; 20 mM 0-dianisidine; 0-dianosidine; 0.2% Triton X-100; 0.5 M Tris-HC1 buffer, pH 7.0) and the hexokinase-glucose-6-phosphate dehydrogenase method ac- cording to the modified procedure of Bergmeyer et al. (11) (hexoki- nase, 1,250 units/l; glucose-6-phosphate dehydrogenase, 1,250 units/ 1; 25 mM ATP, 2.5 mM NADP, 50 mM MgCI,, 50 mM Tris-HC1, pH 7.4). The two analytical methods were required because factors pre- sent in serum interfere with the glucose oxidase/peroxidase method which, therefore, cannot be used with impure samples. The potency (1 unit) of the inhibitor against trehalase activity is defined as the amount of inhibitor that reduces the hydrolysis of trehalose by 1 nmol under the assay conditions described above.

Protein Determination and Characterization-Protein was deter- mined by the method of Bradford (12) using the Bio-Rad protein assay kit with bovine serum albumin as standard.

Amino acid analysis was achieved using a Hitachi 835 automatic analyzer following hydrolysis of the purified protein in 6 M HCl at 110 "C for 24 h.

The NHn-terminal amino acid sequence of purified trehalase inhib- itor was analyzed by automated Edman degradation with a protein sequencer (model 740.4; Applied Biosystems) (13). Samples of purified trehalase inhibitor were prepared for SDS-polyacrylamide gel electro- phoresis by incubation with 80 mM Tris-HC1 buffer (pH 8.8) contain- ing 1% SDS, 1% mercaptoethanol, 30% glycerol, and 0.01% brom- phenol blue in boiling water for 5 min (14). Staining with periodic acid-Schiff reagent was accomplished according to the method of Glossmann and Neville (15).

RESULTS AND DISCUSSION

Serum prepared from the hemolymph of resting adult cock- roaches contains 30-40 mM trehalose which undergoes slight hydrolysis to glucose when the serum is incubated at 30 "C. However, treatment of the serum with trypsin causes a marked increase in the rate of trehalose hydrolysis with a 10- fold elevation of glucose production evident after 90 min of incubation (Fig. 1). The data presented in Fig. 1 indicate also that trypsinization of purified hemocyte trehalase effects only a 20% reduction in enzyme activity, thereby indicating that

0 I

/ * /

0 30 60 90 T i m e (min )

FIG. 1. Effect of trypsinization on glucose production in incubated serum of P. arnericana. Serum was prepared from a resting cockroach and incubated immediately at 30 "C. Trypsinization was achieved according to previously described procedures (9). At each time point, aliquots (10 pl) of serum (circles) and incubation medium containing hemocyte trehalase (triangles) were collected for determination of glucose concentration. Closed symbols indicate tryp- sinized samples.

the enzyme is only partially degraded by the trypsin treat- ment. The results confirm the finding that an inactive form of serum trehalase is activated by trypsinization (9) and support suggestions that an inhibitor of trehalase is associated with the enzyme in the hemolymph of some insects (8, 16). In the light of these observations, studies were undertaken to demonstrate and isolate a trehalase inhibitor in hemolymph.

Injection of adult cockroaches with physiological concen- trations of taurine reduces serum trehalase activity (9); there- fore, the serum from taurine-treated cockroaches was used as a likely source of the trehalase inhibitor. Inhibitory activity against trehalase was obtained by dialyzing the serum from taurine-treated insects against double-distilled water for 15- 20 h and centrifuging the resulting turbid dialysate to yield the inhibitor-containing supernatant fraction.

The supernatant of the dialysate was concentrated by ly- ophilization and applied to gel permeation chromatography on Superose 6 HR 10/30 (Pharmacia LKB Biotechnology Inc.) equilibrated with 250 mM phosphate buffer (pH 6.6). Three major peaks were eluted with the same buffer at a flow rate of 400 pllmin, and as indicated in Fig. 2, the third peak contained the inhibitory activity.

This fraction was further resolved by a reversed phase C,/

0 0 20 40 80

FIG. 2. Gel filtration on Superose 6 (Pharmacia) of the su- pernatant obtained following dialysis of serum. Solvent (250 mM phosphate buffer, pH 6.6; sample injected at time 0). Horizontal bar indicates fractions that show inhibitory activity against trehalase.

T i m e ( m i n 1

0 0 i o 20 30 LO 50 60

T l m e ( r n i n l

on Pro RPC HR5/10 (Pharmacia) of Superose 6-active frac- FIG. 3. Reversed phase fast protein liquid chromatography

tions. Dotted line indicates concentration of CH&N in 0.1% CF,COOH/H,O. Arrow indicates the peak that contains inhibitory activity against trehalase.

Purification of Trehalase Inhibitor from Insect Hemolymph 16167

C8 column (Pro RPC HR 5/10, Pharmacia LKB Biotechnol- ogy Inc.) using gradient elution from 0 to 60% CH&N in 0.1% CF3COOH/Hz0 a t a flow rate of 500 pl/min (Fig. 3). The inhibitory activity was eluted in a single peak which was collected and rechromatographed using a large-pore C8-deriv- atized silica HPLC column (Yamamura Co., Japan, 4.6 mm inner diameter X 250 mm) under two elution schedules. The active fraction was first eluted using a gradient of CH&N from 36 to 42% in 0.156 CF3COOH/H20 at a flow rate of 1 ml/min (Fig. 4a) and thsen reapplied to the column and eluted with a gradient of 3749% CH&N in 0.1% CF3COOH/H20 at a flow rate of 1 ml/min (Fig. 4b). Data on the purification procedure are presented. in Table I and demonstrate 77.6-fold purification with a 17% yield.

SDS-polyacrylamide gel electrophoresis of the purified tre- halase inhibitor yielded a single band which showed a positive glucoprotein pattern with periodic acid-Schiff reagent (Fig. 5). Comparison of the electrophoretic mobility of the protein with that of standard proteins indicates a molecular weight of about 86,000 (Fig. 5).

The amino acid composition of the purified inhibitor is

0.1

E 0 w - U 2 0.05 C 0 ; n Q

0

0.1

E

w i3

0

2 0.0: C 0 2 Lo n Q

0

I

0 10 20 30 40 50 T i m e ( m i n )

FIG. 4. HPLC on Cls column (pore size, 300 A). a, Pro RPC HR 5/10-active fractions chromatographed with CH&N in 0.1% CF&OOH/HzO; b, C8-actiwe fractions rechromatographed with the same solvents using a smaller gradient than in a. Dotted line indicates concentration of solvent. Arrow indicates peak that contains inhibi- tory activity against trehalase.

shown in Table I1 and the NH2-terminal amino acid sequence for the first 19 residues is given in Fig. 6. The amino acid occurring at position 11 was not identified. Sequence compar- ison was made against the National Biomedical Research Foundation Protein Data Bank using Beckman’s MicroGenie version 4.0, but no major homologies were found.

The purified inhibitor shows a dose-dependent capacity to inhibit trehalase purified from cockroach hemocytes with an IC6,, of 5.4 pg under the assay conditions employed in the study (Fig. 7). The linear decrease of activity of trehalase with increasing concentration of the inhibitor implies strong com- petitive binding. This interpretation is supported by the result of the Ackermann-Potter plot presented in Fig. 8. At low concentrations, all of the enzyme exists as an enzyme-inhib- itor ( [ E . I]) complex which does not exhibit activity, but the slope of the plot is proportional to enzyme concentration when the concentration exceeds that of the inhibitor. This result indicates that the inhibitor competes with the substrate for the catalytic site, and then the enzyme in the E.1 complex will not contribute to the initial enzymic reaction velocity because it dissociates slowly (17).

The trehalase activity to cockroach hemolymph increases markedly in response to exercise (6) and is greater in insects that are in an excited state than in those that have been isolated for several hours in individual Petri dishes (4). How- ever, the mechanism by which the trehalase activity increases under these circumstances has not been elucidated. The dem- onstration that treatment of serum from resting cockroaches with trypsin increases endogenous hydrolysis of trehalose (9, Fig. 1) suggests that under resting conditions, serum trehalase is inhibited through association with a protein/peptide inhib- itor. The isolation and partial characterization of a serum protein (Mr 86,000) which strongly inhibits purified trehalase, as reported in the present study, provides compelling support for this proposal. The data indicate that activation of serum trehalase may involve cleavage of the enzyme-inhibitor com- plex.

Trehalase activity occurs also in the hemocyte component of whole hemolymph (5), and, indeed, this compartmentali- zation of the enzyme has been invoked to account for the co- existence of trehalose and trehalase in hemolymph (2). In “excited insects, the trehalase content of serum exceeds that of the whole hemolymph from which the serum fraction is derived, probably as a result of the release and activation of trehalase from hemocytes during the collection of serum (4). Thus, the increased serum trehalase activity in excited insects may be due to the removal of inhibitor protein from endoge- nous serum trehalase and newly released hemocyte trehalase.

Hemolymph taurine levels increase following the onset of exercise and remain elevated during the post-exercise decline

TABLE I Summary of purification of trehalase inhibitor from serum of the American cockroach

Total volume Protein Total Specific activity activity

ml Mf ml units units f@ -fold 7%

Purification” Recovery

Serum 25.5‘ 82,700 Supernatant after dialysis 27.3 3,520 Superose 6 36.2 196 Pro RPC

948.4 0.134 12.1 25.0

1 100

C8 1 451.3

4.2 1.49 11.1

15.6 48

cs I1 260.4 3.97

2.1 7.3 160.0 29.7 27

10.4 77.6 17

- - - - - - - -

’ Purification and recovery are based on the specific activity and total activity, respectively, of the Superose 6

* The volume of serum collected from about 350 cockroaches. fraction.

Serum artd supernatant after dialysis contained too much trehalase to permit detection of inhibitory activity.

16168 Purification of Trehalase Inhibitor from Insect Hemolymph

4

kd C B B P A S

200 116 92.5

66.2

4 s

Standanl t t+- Sample Stan& " rd

FIG. 5. SDS-polyacrylamide gel electrophoresjs of purified trehalase inhibitor. About 1.5 pg of protein was subjected to SDS- polyacrylamide gel electrophoresis and right and left halves of the gels stained with Coomassie Blue (CBB) and periodic acid-Schiff (PAS), respectively. The sample indicated is purified inhibitor.

TABLE I1 Amino acid composition of trehalase inhibitor

Values were obtained from a sinale analvsis. ~~

mo1/1000 mol Asx 123 Thr 63 Ser 61 Glx 98 Pro 58 G1Y 104 Ala 92 CYS 16 Val 69 Met 4 Ile 33

Leu 79

Phe 35 His 32

TYr 38

LYS 47 -4% 48

(TW) - Tryptophan was decomposed during acid hydrolysis.

of trehalase activity (9). Furthermore, as injection of physio- logical levels of taurine results in trehalase inhibition, it has been suggested that taurine plays an indirect role in regulating trehalase activity (9). Taurine also reduces 45Ca2+ accumula- tion in resting and depolarized locust synaptosomes (18) and decreases octopamine-stimulated CAMP production in cock- roach hemocytes (19). Octopamine also causes increased Ca2+ accumulation in an insect hemocyte cell line (20), and it is possible that taurine inhibits this effect. Given that octopa- mine serves a sympathetomimetic function in insects and that hemolymph octopamine levels rise rapidly in response to excitation (21), it is reasonable to propose that octopamine acts directly on hemocytes to effect release of trehalase. An octopamine-mediated elevation of hemolymph trehalase has been reported (22). It is reasonable also to propose that the inhibitory action of taurine on hemolymph trehalase is ex- pressed through inhibition of the Ca2'-mediated transmem- brane signaling trehalase from hemocytes. This hypothesis is further supported by the observation that treatment of cock- roaches with EDTA inhibits hemolymph trehalase activity,

1 5 H - A l a - I l e - Pro - Thr - Pro - H i s - V a l - Tyr - L y s -

1 0 V a l - ? - V a l - Pro - A s p - G l y - A l a - L e u - A s n - A s p -

15

FIG. 6. NHS-terminal amino acid sequence of trehalase in- hibitor.

\

0 ' 0 2 4 6 8 1 0

I n h i b i t o r ( p g )

FIG. 7. Effect of various concentrations of purified inhibi- tor protein on trehalase activity. Trehalase concentration was 63 pg protein/ml, and the amount of added inhibitor varied according to the levels indicated on the abcissa.

0

0 2.5 5.0 T r e h a l a s e (pg/rnl)

FIG. 8. Ackermann-Potter plot of trehalase preincubated with purified inhibitor protein. Various amounts of trehalase as indicated on the abcissa were preincubated with a fixed concentration of inhibitor for 15 min at 30 "C to establish equilibrium. The enzyme reaction was started by adding trehalose and the hexokinase-glucose- 6-phosphate dehydrogenase reagent (see "Materials and Methods") and then followed by measuring the increase in absorbancy a t 340 nm at 30 "C. The concentration of purified inhibitor protein used were as follows: A, 111 nm, 0, 74.4 nM; 0, 0 nM.

and this inhibition can be removed by administration of bivalent cations (5).

The current study does not explain the mechanism by which the inhibitor protein is cleaved from the native enzyme during excitation or the mechanism by which the normal state of trehalase inhibition is restored. These processes are currently under study.

Acknowledgment-The valued technical assistance of David C. Watson is gratefully appreciated.

REFERENCES

1. Wyatt, G. R. (1967) Adu. Insect Physiol. 4, 287-360 2. Katagiri, C. (1977) Insect Biochem. 7, 351-353 3. Van Handel, E. (1978) J. Insect Physiol. 24 , 151-153 4. Downer, R. G. H., and Matthews, J. R. (1978) Can. J. Zool. 56,

5. Matthews, J. R., Downer, R. G. H., and Morrison, P. E. (1976)

6. Downer, R. G . H., and Matthews, J. R. (1977) J. Insect Physiol.

2217-2219

J. Insect Physiol. 22, 157-163

23,1429-1435

Purification of Trehalase Inhibitor from Insect Hemolymph 16169

7. Downer, R. G. H. (1.981) in Energy Metabolism in Insects 15. Glossmann, H., and Neville, D. M., Jr. (1971) J. Biol. Chem. 246, (Downer, R. G. H., ed) pp. 1-17, Plenum Publishing Corp., 6339-6346 New York 16. Candio, L. M., Marechal, L. R., and Veiga, L. A. (1981) Archos.

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H. (1987) FEBS Lett. 219,83-87 Methods of Enzymatic Analysis (Williamson, D. H., ed) vol- 3, 20. Jahagirdar, A. p., Milton, G., Viswanatha, T., and Downer, R. G. pp. 1196-1201, Academic Press, Orlando, FL

12. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 13. Hewick, R. H., Hunkapiller, M. W., Hood, L. E., and Dreyer, W.

14. Laemmli, U. K. (1970) Nature 227,680-685 Biochim. Biophys. Acta 801, 177-183

21. Bailey, B. A., Martin, R. J., and Downer, R. G. H. (1984) Can. J.

22. Jahagirdar, A. P., Downer, R. G. H., and Viswanatha, T. (1984) J. (1981) J. Biol. Chcnm. 256, 7990-7997 2001. 62, 19-22