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α-d -Glucosidase in the midgut of the american cockroach, Periplaneta Americana
Insect Biochem., Vol. 9, pp. 205 to 209. Pergamon ~Press Ltd. 1979. Printed in Great Britain.
0020-1700/79/0301-0205 $02.00/0
7-D-GLUCOSIDASE IN THE MIDGUT OF THE AMERICAN COCKROACH, P E R I P L A N E T A A M E R I C A N A CHIHIRO KATAGIRI Biochemistry Laboratory, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan (Received 12 June 1978) A ~ t r a c t - - A sugar-hydrolyzing enzyme was purified from the midgut of the American cockroach by DEAEcellulose and affinity column chromatography. Examination of its substrate specificity showed that this enzyme is an :t-D-glucosidase (E.C.3.2.1.20). The purified enzyme preparation exhibited a high degree of homogeneity on an acrylamide gel. However, it appeared that the preparation was still contaminated with other glycosidases such as trehalase (E.C.3.2.1.28), fl-D-glucosidase (E.C.3.2.1.21) or :t-amylase (E.C.3.2.1. I). The isoelectric point of the enzyme is very low, pH 3.54, the pH optimum is 5.8 and the K,, value is 5.0 mM for p-nitrophenyl-:~-D-glucopyranoside. These characteristics show that the midgut :t-Dglucosidase is distinct from the serum of ~-o-glucosidase. Key Word Index: American cockroach, midgut, :t-D-glucosidase
specificity was determined by gas-liquid chromatography (GLC). Ten #1 of enzyme solution was incubated with 100/tl AN 0~-D-GLUCOSIDASE(E.C.3.2.1.20) in the serum o f the of the substrate dissolved in 0.2 M phosphate buffer (pH 6.0) at 30°C for 2 hr. The following substrates were used: 5 mM A m e r i c a n cockroach has been purified a n d characterized (KATAGIRI, 1979) a n d 0t-o-glucosidase trehalose, maltose, sucrose, turanose, cellobiose, lactose, (maltase) h a s been reported previously in the maltotriose, melezitose and raffinose (all from Nakarai alimentary canal o f this insect (WIGGLESWORTH, Chemicals), and 0.5~ soluble starch (Wako Pure Chemical Industries). After incubation, the reaction was stopped by 1927). A l t h o u g h there is an extensive literature on the placing the tubes in boiling water for 5 min. The sample was purification a n d p r e p a r a t i o n o f m a m m a l i a n intestinal subjected to GLC analysis to determine the monosaccharides ct-glucosidase (e.g. SEMENZA, 1976; WILSON, 1978), liberated as described previously (KATAGIR1,1977). very little work o f any kind has been d o n e with insect Enzyme pur~[ication 0~-D-glucosidase. Recently, this enzyme (sucrase) w a s Before preparation of the enzyme, each cockroach was purified from Drosophila (MARZLUF, 1969; HUBER a n d transferred from the colony to a separate petri dish and LEFEBVRE, 1971) a n d h o n e y bee (HUBER, 1975) b u t in starved for 3 days. During this period the gut became almost all cases enzyme was isolated from the whole body or empty of food. The animals were placed on ice and the a b d o m e n . Since ct-o-glucosidase was f o u n d b o t h in midgut was dissected free of adhering tissues. The midgut was m i d g u t a n d in serum o f the A m e r i c a n cockroach, cut longitudinally and thoroughly rinsed with ice-cold 0.15 M purification o f 7-o-glucosidase for m i d g u t m a y be KCI to remove the lumen contents as completely as possible. helpful in c o m p a r i n g the physiological roles o f the two The midguts were stored at - 2 0 ° C until use. Twenty to enzymes. T h e present paper is the first report o f studies twenty-five midguts were homogenized in 5 ml of ice-cold on purification o f ~t-D-glucosidase from the m i d g u t distilled water with a Virtis 45 homogenizer. The homogenate was filtered through filter paper, and the filtrate centrifuged a n d on c o m p a r i s o n between midgut a n d serum ~-oat 3000 g for 15 rain. More than 90% of the enzyme activity glucosidases. was found in the supernatant, which was then dialyzed against 0.05 M phosphate buffer (pH 6.0). The dialyzed MATERIALS AND METHODS sample was applied to a DEAE-cellulose column (1.4 cm i.d. x 45 cm) equilibrated with 0.05 M phosphate buffer (pH Animals 6.0). After washing the column with 200 ml of this buffer, the Male adults of Periplaneta americana fed on dog biscuits enzyme was eluted with 300 ml of a linear gradient of KCI and water ad libitum were used throughout the experiments. from 0 to 0.6 M in the same buffer (flow rate, 10 ml/hr). The fractions were collected and dialyzed against 0.02 M Enzyme assay phosphate buffer (pH 6.0). For the further purification, the The method involved measurement of the rate of dialyzed solution (35 #g protein/ml) was subjected to an hydrolysis of p-nitrophenyl-a-D-glucopyranoside (PNPG) affinity column (0.9 cm i.d. x 15 cm) equilibrated with 0.02 (Nakarai Chemicals), using a spectrophotometric assay of M phosphate buffer (pH 6.0). The adsorbent for the affinity the p-nitrophenol released by enzyme action. The reaction column chromatography (supplied by Dr. T. Mega of Osaka mixture contained 10/~1 of enzyme solution and 20 #1 of 20 University) was prepared by coupling di-e-aminocaproyl-pmM PNPG in 250 ~1 of 0.2 M phosphate buffer (pH 6.0). The aminophenyl-fl-o-giucopyranoside with CNBr-activated mixture was incubated at 30°C for 15 min. The reaction was Sepharose 4B (MEGAand MA1"SUSHIMA,1977). The enzyme stopped by addition of 500 ~1 of a saturated solution of was eluted by a stepwise change of the following ionic NazCO 3 and the absorbance was measured at 400 nm. The strength: 100 ml of 0.02, 0.03, 0.05, 0. l and 0.2 M phosphate activity of enzyme equivalent to hydrolyzing 1 #mole of buffer (pH 6.0) and 0.3 M KCl in 0.2 M phosphate buffer (pH PNPG during 15 min was defined as one unit. Substrate 6.0). The flow rate was l0 ml/hr. 205 INTRODUCTION
CI-III-IlROKA'fAGIRI
206
Table l. Purification of 2-D-glucosidase from American cockroach midgut Step Crude extract (supernatant of homogenate) DEAE-cellulose column chromatography Affinity column chromatogaphy
Protein (mg)
Activity (units*)
102.9
4160
28.2 6.86
. Specific activity (units/mg protein)
Purification (fold)
Yield ('~o)
40
1.0
100
4070
144
3.6
98
2030
296
7.4
49
* See Materials and Methods for definition of unit.
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was run on 7.5~o gels according to DAvis (1964). After the run, the gels were incubated with 10 mM PNPG in 0.2 M phosphate buffer (pH 6.0) at 30°C. The enzyme was located by a yellow band resulting from the liberation of p-nitrophenol. The yellow band was scanned at 400 nm using a Shimadzu DualWavelength Chromatoscanner, CS-900. After the scan, the gel was further stained with Coomassie brilliant blue R to locate all protein bands on disc gels. These protein bands were also scanned at 550 nm.
elution profile of the enzyme from the DEAE-cellulosc column. The ~-D-glucosidase was eluted with a linear gradient of KCI and the activity appeared in a single peak. The combined fractions collected (Nos. 181-210) were applied to affinity column chromatography and, as shown in Fig. 2, when the column was run with 0.1 M phosphate buffer, most of the enzyme (89% of total activity, fraction I) was eluted. A second minor fraction (fraction II) also appeared when run with 0.2 M buffer. Fraction 1 coincided with a small protein peak.
Isoelectricfocusing lsoelectric focusing of purified enzyme was performed on a LKB-8100-1 electrofocusing column (110 ml) at I°C with Ampholine in a glycerol gradient fiom 70% (w/v). The concentration of Ampholine was 1% (w/v) for pH 2.5-10 or 0.5~o (w/v) for pH 2.5-4. Focusing was attained in 18 hr, applying an initial voltage of 1400 V and a final voltage of 1600 V. RESULTS
Homogeneity of the enzyme preparation The homogeneity of the above fraction I was tested by polyacrylamide gel electrophoresis; a single sharp peak of protein appeared near the front, and coincided with a sharp peak of the enzyme activity (Fig. 3). A similar test of fraction II showed essentially the same profile as fraction I, with the two fractions having the same mobility.
Purification of enzyme Table 1 shows the procedure for purification on ~-Dglucosidase from the midgut of the American cockroach. The procedure resulted in a purification of 7.4-fold with a specific activity of 296 units/rag protein. The recovery was 49%. Figure 1 shows the
Isoelectric point The isoelectric point of the enzyme was estimated by electrofocusing of fraction I at the pH ranges 2.5-10 and 2.5-4. A single sharp peak of enzyme activity was observed at pH 3.54 (Fig. 4).
! 1
300 0
1.0
~ 2oc
:-~ ~o0 .,.,..,
0 50
100
150
200
Fraction No. ( 2 m l / f r a c t i o n
250
)
Fig. l. Elution profile of ~-D-glucosidase on DEAE-cellulose column. A column (1.4 cm i.d. x 45 cm) was eluted with 300 ml of a linear gradient of 0-0.6 M KCI in 0.05 M phosphate buffer (pH 6.0). O O Enzyme activity, H absorbance at 280 nm.
:~-D-Glucosidasein cockroach midgut
207
I--
.9 o~ O
L(3 O
0 ,L
5C ~o.
0 J,
¢~3 "7.
¢~
o I ~
4O
6 .L
6 ,L
0.3
I
E 0-2 ¢
30
>
O
oO ¢N
u
20 10
50
100 150 200 Fraction No. ( 2 rnl I fraction )
250
300
Fig, 2. Elution profile of ~-D-glucosidaseon the affinity column chromatography. Thirty 1-ml fractions (Nos. 181-210 from the DEAE-cellulosecolumn), containing 1.1 mg protein, was applied to Sepharose 4B derivative substituted with di-e-aminocaproyl-p-aminophenyl-fl-D-glucopyranoside (column 0.9 cm i.d. x 15 cm). See the text for details. O------O Enzyme activity, O-----O absorbance at 280 nm.
Effect of temperature. The thermal stability of the ~D-glucosidase was examined between 0 and 80°C. As The properties of the enzyme were tested using the enzyme (fraction I) from the affinity chromatography. shown in Fig. 7, the enzyme was stable to 50°C, but Michaelis constant (Kin). Figure 5 shows a became partially denatured at temperatures beyond 50°C; the incubation at 60°C for 5 min caused 70% loss Lineweaver-Burk plot with PNPG as substrate. The Kmvalue for PNPG was 5.0 mM in 0.2 M phosphate of activity, and the enzyme activity was completely lost at 65°C. buffer (pH 6.0). Substrate specificity. Various sugars were incubated pH Optimum. The effect of pH on the ~t-Dglucosidase activity was tested using 0.2 M citrate with the enzyme (fraction I) to test the substrate specificity, and the liberated products were analyzed buffer (pH 4.0-5.9) and 0.2 M phosphate buffer (pH 5.7-8.0). Figure 6 demonstrates that the pH optimum by GLC. The data given in Table 2 show that the enzyme preparation splits ~-D-glucosides including was 5.8 when either buffer system was used. trehalose and starch, and also a fl-o-glucoside (cellobiose), but not a fl-o-galactoside (lactose) or the trisaccharide raffinose. Nature of ~-o-glucosidase
®->
® 7
1 .=
6 5
u E
E
=4
z3
v
"3
I=-
o=
2 1 J
..... 0
/
L
/
\
i
i
i
i
i
1
2
3
4
5
100
%
(cm) Fig. 3. Polyacrylamidegel electrophoresisof the fraction I of the affinity column, protein (Coomassie stain); . . . . . :tn-glucosidase activity• I.B. 9 / 2 - - F
L ......... Jo
50 Fraction No. ( 1 roll f r . )
Fig. 4. Isoelectricfocusing of the fraction I in a pH gradient 2.5-4 after 18-hrrun. Anode solution, 0.2 M phosphoric acid; cathode solution, 0.25 M NaOH. H Enzyme activity, O----O pH gradient.
f/
208
CHIHIRO KATAGIRI
10C
50
1
"5 rr o, o
0
2'0
4'0 5'0
60 70 80
Temperature ( ° C )
/ 0
Fig. 7. Effect of temperature on zt-D-glucosidase. The enzyme was incubated at various temperatures for 5 rain, and immediately placed at 30°C before assay of enzyme activity. Each point is the mean + S.E. of four experiments.
IIS ( mM -1 )
Fig. 5. Lineweaver-Burk plots of purified :~-D-glucosidase with various concentrations of PNPG.
DISCUSSION Polyacrylamide gel electrophoresis (Fig. 3) of fraction I shows the high homogeneity of the enzyme preparation and, since fraction I can hydrolyze melezitose but not act on raffinose, the presence of an ~-D-glucosidase in this fraction is evident. However, its substrate specificity suggested that it was contaminated with glycosidases other than s-Oglucosidase, for example, with trehalase (E.C.3.2.1.28), fl-D-glucosidase (E.C.3.2.1.21) and 7amylase (E.C.3.2.1.1). This contamination is to be expected as the adsorbent used for affinity
100
l\
/
v
chromatography contains a fl-glucosidic residue as a ligand and this is known not to be specific for ~-oglucosidase (MEGA and MATSUSHIMA, 1977). To eliminate such possible contaminants, further gel filtration was attempted, using Sephadex G-100 or Bio-Gel P-200. However, s~-o-glucosidase was always accompanied by other glycosidases, and appeared with the void volume of these columns. This may be explained by a possible formation o f a large complex consisti,ag of ~-D-glucosidase and other contaminants during the passage through such gels. Evidence has been obtained that :~-D-glucosidase exists in the serum of the American cockroach (KATAGIRI, 1979). It is interesting to compare the roidgut 7-D-glucosidase with the serum 7-oglucosidase. The K,, values are 5.0 and 2.8 mM, and p H optima 5.8 and 6.8, for the midgut enzyme and serum enzyme, respectively. The midgut enzyme is more heat-stable than the serum enzyme. These two enzymes exhibit different elution profiles on D E A E cellulose and affinity column chromatography, the serum enzyme is more easily eluted from the columns with lower concentration of the buffer. Also, the migration patterns of the two enzymes on polyacrylamide gels are different; the midgut enzyme migrates much faster than that of the serum, indicating
._> .5 5O
Table 2. Substrate specificity of glycosidases of fraction I
.>_-,
Sugar
(Structure)
Treha|ose Maltose Sucrose Turanose Cellobiose Lactose Maltotriose Melezitose Ra.ffinose Soluble starch
(Glc71 --*~1Glc)* (Glc~ 1~4Glc) (Glc~ 1~fl2Fru) (Glc~ 1--*3Fru) (Glcfll ---,4Glc) (Galfll ~4Glc) (Glca 1---,4G1c71--*4Glc) (Glc~ 1~ 3Frufl2---,~ 1Glc) (Gal~ 1--*6Glca 1-+f12Fru)
~aJ =_ 4
,
5
6 pH
7
B
Fig. 6. Effect of pH on ~-D-glucosidase. The enzyme was incubated with PNPG at different pH values for 15 rain at 30°C. H Citrate buffer, O O phosphate buffer. • Each point is the mean ___ S.E: of four experiments.
-
Hydrolysis + + + + + + + +
* Gal---galactose; Glc---glucose; Fru --fructose; + positive negative.
~t-o-Glucosidase in cockroach midgut Table 3. ~-D-Glucosidase in haemolymph of different insects Insects Lepidoptera Philosamia cynthia Bombyx mori Galleria mellonella Coleoptera Tenebrio molitor Hymenoptera Trichiocampus populi Diptera Sarcophaga peregrina
Stage Pupa Larva Larva
Activity
R
m
Adult Larva Larva
+ Present: - absent. t h a t the midgut enzyme is more acidic. In fact, Fig. 4 clearly d e m o n s t r a t e s t h a t isoelectric point of the enzyme is very low, p H 3.54, which c a n explain the a p p e a r a n c e of enzyme b a n d near the front o n polyacrylamide gel (Fig. 3). All the a b o v e d a t a suggests t h a t the midgut enzyme a n d the serum enzyme are different enzymes. Acknowledgements--I wish to thank Professor H. CHINO for his helpful discussions and advice and Dr. R. G. H. DOWNER of the University of Waterloo for critically reading the manuscript. This study was supported in part by research grants from the Ministry of Education of Japan and from Toray Foundation.
209
REFERENCES
DAVISB. J. (1964) Disc electrophoresis--lI. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121, 404--427. HUBERR. E. (1975) The purification and study of a honey bee abdominal sucrase exhibiting unusual solubility and kinetic properties. Archs Biochem. Biophys. 168, 198-209. HUBER R. E. and LEFEBVREY. A. ( 1971 ) The purification and some properties of soluble trehalase and sucrase from Drosophila melanogaster. Can. J. Biochem. 49, I 155-1164. KATAGIRI C. (1977) Localization of trehalase in the haemolymph of the American cockroach, Periplaneta americana. Insect Biochem 7, 351-353. KATAGIRI C. (1979) :t-D-Glucosidase in the serum of the American cockroach, Periplaneta americana. Insect Biochem. 9, 199-104. MARZLUF G. A. (1969) Studies of trehalase and sucrose of Drosophila melanogaster Archs Biochem Biophys, 134, 8-18. MEGA T. and MATSUSHIMA Y. (1977) Affinity chromatography of glycosidases--II. Studies on specific and non-specific binding. J. Biochem.81,571-578. SEMENZA G. (1976) Glucosidase of small intestinal brush border. In Membrane and Disease (Ed. by BOLLS L., HOFFMANN J. F. and LEAF A.), pp. 243-252. Ra',,~ii Fress, New York. WIGGLESWORTH V. B. (1927) CVI digestion in the cockroach--II. The digestion of carbohydrates. Biochem J. 21, 797-811. WILSON D. B. (1978) Cellular transport mechanisms. Ann Rev. Biochem. 47, 933-966.
0020-1700/79/0301-0205 $02.00/0
7-D-GLUCOSIDASE IN THE MIDGUT OF THE AMERICAN COCKROACH, P E R I P L A N E T A A M E R I C A N A CHIHIRO KATAGIRI Biochemistry Laboratory, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan (Received 12 June 1978) A ~ t r a c t - - A sugar-hydrolyzing enzyme was purified from the midgut of the American cockroach by DEAEcellulose and affinity column chromatography. Examination of its substrate specificity showed that this enzyme is an :t-D-glucosidase (E.C.3.2.1.20). The purified enzyme preparation exhibited a high degree of homogeneity on an acrylamide gel. However, it appeared that the preparation was still contaminated with other glycosidases such as trehalase (E.C.3.2.1.28), fl-D-glucosidase (E.C.3.2.1.21) or :t-amylase (E.C.3.2.1. I). The isoelectric point of the enzyme is very low, pH 3.54, the pH optimum is 5.8 and the K,, value is 5.0 mM for p-nitrophenyl-:~-D-glucopyranoside. These characteristics show that the midgut :t-Dglucosidase is distinct from the serum of ~-o-glucosidase. Key Word Index: American cockroach, midgut, :t-D-glucosidase
specificity was determined by gas-liquid chromatography (GLC). Ten #1 of enzyme solution was incubated with 100/tl AN 0~-D-GLUCOSIDASE(E.C.3.2.1.20) in the serum o f the of the substrate dissolved in 0.2 M phosphate buffer (pH 6.0) at 30°C for 2 hr. The following substrates were used: 5 mM A m e r i c a n cockroach has been purified a n d characterized (KATAGIRI, 1979) a n d 0t-o-glucosidase trehalose, maltose, sucrose, turanose, cellobiose, lactose, (maltase) h a s been reported previously in the maltotriose, melezitose and raffinose (all from Nakarai alimentary canal o f this insect (WIGGLESWORTH, Chemicals), and 0.5~ soluble starch (Wako Pure Chemical Industries). After incubation, the reaction was stopped by 1927). A l t h o u g h there is an extensive literature on the placing the tubes in boiling water for 5 min. The sample was purification a n d p r e p a r a t i o n o f m a m m a l i a n intestinal subjected to GLC analysis to determine the monosaccharides ct-glucosidase (e.g. SEMENZA, 1976; WILSON, 1978), liberated as described previously (KATAGIR1,1977). very little work o f any kind has been d o n e with insect Enzyme pur~[ication 0~-D-glucosidase. Recently, this enzyme (sucrase) w a s Before preparation of the enzyme, each cockroach was purified from Drosophila (MARZLUF, 1969; HUBER a n d transferred from the colony to a separate petri dish and LEFEBVRE, 1971) a n d h o n e y bee (HUBER, 1975) b u t in starved for 3 days. During this period the gut became almost all cases enzyme was isolated from the whole body or empty of food. The animals were placed on ice and the a b d o m e n . Since ct-o-glucosidase was f o u n d b o t h in midgut was dissected free of adhering tissues. The midgut was m i d g u t a n d in serum o f the A m e r i c a n cockroach, cut longitudinally and thoroughly rinsed with ice-cold 0.15 M purification o f 7-o-glucosidase for m i d g u t m a y be KCI to remove the lumen contents as completely as possible. helpful in c o m p a r i n g the physiological roles o f the two The midguts were stored at - 2 0 ° C until use. Twenty to enzymes. T h e present paper is the first report o f studies twenty-five midguts were homogenized in 5 ml of ice-cold on purification o f ~t-D-glucosidase from the m i d g u t distilled water with a Virtis 45 homogenizer. The homogenate was filtered through filter paper, and the filtrate centrifuged a n d on c o m p a r i s o n between midgut a n d serum ~-oat 3000 g for 15 rain. More than 90% of the enzyme activity glucosidases. was found in the supernatant, which was then dialyzed against 0.05 M phosphate buffer (pH 6.0). The dialyzed MATERIALS AND METHODS sample was applied to a DEAE-cellulose column (1.4 cm i.d. x 45 cm) equilibrated with 0.05 M phosphate buffer (pH Animals 6.0). After washing the column with 200 ml of this buffer, the Male adults of Periplaneta americana fed on dog biscuits enzyme was eluted with 300 ml of a linear gradient of KCI and water ad libitum were used throughout the experiments. from 0 to 0.6 M in the same buffer (flow rate, 10 ml/hr). The fractions were collected and dialyzed against 0.02 M Enzyme assay phosphate buffer (pH 6.0). For the further purification, the The method involved measurement of the rate of dialyzed solution (35 #g protein/ml) was subjected to an hydrolysis of p-nitrophenyl-a-D-glucopyranoside (PNPG) affinity column (0.9 cm i.d. x 15 cm) equilibrated with 0.02 (Nakarai Chemicals), using a spectrophotometric assay of M phosphate buffer (pH 6.0). The adsorbent for the affinity the p-nitrophenol released by enzyme action. The reaction column chromatography (supplied by Dr. T. Mega of Osaka mixture contained 10/~1 of enzyme solution and 20 #1 of 20 University) was prepared by coupling di-e-aminocaproyl-pmM PNPG in 250 ~1 of 0.2 M phosphate buffer (pH 6.0). The aminophenyl-fl-o-giucopyranoside with CNBr-activated mixture was incubated at 30°C for 15 min. The reaction was Sepharose 4B (MEGAand MA1"SUSHIMA,1977). The enzyme stopped by addition of 500 ~1 of a saturated solution of was eluted by a stepwise change of the following ionic NazCO 3 and the absorbance was measured at 400 nm. The strength: 100 ml of 0.02, 0.03, 0.05, 0. l and 0.2 M phosphate activity of enzyme equivalent to hydrolyzing 1 #mole of buffer (pH 6.0) and 0.3 M KCl in 0.2 M phosphate buffer (pH PNPG during 15 min was defined as one unit. Substrate 6.0). The flow rate was l0 ml/hr. 205 INTRODUCTION
CI-III-IlROKA'fAGIRI
206
Table l. Purification of 2-D-glucosidase from American cockroach midgut Step Crude extract (supernatant of homogenate) DEAE-cellulose column chromatography Affinity column chromatogaphy
Protein (mg)
Activity (units*)
102.9
4160
28.2 6.86
. Specific activity (units/mg protein)
Purification (fold)
Yield ('~o)
40
1.0
100
4070
144
3.6
98
2030
296
7.4
49
* See Materials and Methods for definition of unit.
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was run on 7.5~o gels according to DAvis (1964). After the run, the gels were incubated with 10 mM PNPG in 0.2 M phosphate buffer (pH 6.0) at 30°C. The enzyme was located by a yellow band resulting from the liberation of p-nitrophenol. The yellow band was scanned at 400 nm using a Shimadzu DualWavelength Chromatoscanner, CS-900. After the scan, the gel was further stained with Coomassie brilliant blue R to locate all protein bands on disc gels. These protein bands were also scanned at 550 nm.
elution profile of the enzyme from the DEAE-cellulosc column. The ~-D-glucosidase was eluted with a linear gradient of KCI and the activity appeared in a single peak. The combined fractions collected (Nos. 181-210) were applied to affinity column chromatography and, as shown in Fig. 2, when the column was run with 0.1 M phosphate buffer, most of the enzyme (89% of total activity, fraction I) was eluted. A second minor fraction (fraction II) also appeared when run with 0.2 M buffer. Fraction 1 coincided with a small protein peak.
Isoelectricfocusing lsoelectric focusing of purified enzyme was performed on a LKB-8100-1 electrofocusing column (110 ml) at I°C with Ampholine in a glycerol gradient fiom 70% (w/v). The concentration of Ampholine was 1% (w/v) for pH 2.5-10 or 0.5~o (w/v) for pH 2.5-4. Focusing was attained in 18 hr, applying an initial voltage of 1400 V and a final voltage of 1600 V. RESULTS
Homogeneity of the enzyme preparation The homogeneity of the above fraction I was tested by polyacrylamide gel electrophoresis; a single sharp peak of protein appeared near the front, and coincided with a sharp peak of the enzyme activity (Fig. 3). A similar test of fraction II showed essentially the same profile as fraction I, with the two fractions having the same mobility.
Purification of enzyme Table 1 shows the procedure for purification on ~-Dglucosidase from the midgut of the American cockroach. The procedure resulted in a purification of 7.4-fold with a specific activity of 296 units/rag protein. The recovery was 49%. Figure 1 shows the
Isoelectric point The isoelectric point of the enzyme was estimated by electrofocusing of fraction I at the pH ranges 2.5-10 and 2.5-4. A single sharp peak of enzyme activity was observed at pH 3.54 (Fig. 4).
! 1
300 0
1.0
~ 2oc
:-~ ~o0 .,.,..,
0 50
100
150
200
Fraction No. ( 2 m l / f r a c t i o n
250
)
Fig. l. Elution profile of ~-D-glucosidase on DEAE-cellulose column. A column (1.4 cm i.d. x 45 cm) was eluted with 300 ml of a linear gradient of 0-0.6 M KCI in 0.05 M phosphate buffer (pH 6.0). O O Enzyme activity, H absorbance at 280 nm.
:~-D-Glucosidasein cockroach midgut
207
I--
.9 o~ O
L(3 O
0 ,L
5C ~o.
0 J,
¢~3 "7.
¢~
o I ~
4O
6 .L
6 ,L
0.3
I
E 0-2 ¢
30
>
O
oO ¢N
u
20 10
50
100 150 200 Fraction No. ( 2 rnl I fraction )
250
300
Fig, 2. Elution profile of ~-D-glucosidaseon the affinity column chromatography. Thirty 1-ml fractions (Nos. 181-210 from the DEAE-cellulosecolumn), containing 1.1 mg protein, was applied to Sepharose 4B derivative substituted with di-e-aminocaproyl-p-aminophenyl-fl-D-glucopyranoside (column 0.9 cm i.d. x 15 cm). See the text for details. O------O Enzyme activity, O-----O absorbance at 280 nm.
Effect of temperature. The thermal stability of the ~D-glucosidase was examined between 0 and 80°C. As The properties of the enzyme were tested using the enzyme (fraction I) from the affinity chromatography. shown in Fig. 7, the enzyme was stable to 50°C, but Michaelis constant (Kin). Figure 5 shows a became partially denatured at temperatures beyond 50°C; the incubation at 60°C for 5 min caused 70% loss Lineweaver-Burk plot with PNPG as substrate. The Kmvalue for PNPG was 5.0 mM in 0.2 M phosphate of activity, and the enzyme activity was completely lost at 65°C. buffer (pH 6.0). Substrate specificity. Various sugars were incubated pH Optimum. The effect of pH on the ~t-Dglucosidase activity was tested using 0.2 M citrate with the enzyme (fraction I) to test the substrate specificity, and the liberated products were analyzed buffer (pH 4.0-5.9) and 0.2 M phosphate buffer (pH 5.7-8.0). Figure 6 demonstrates that the pH optimum by GLC. The data given in Table 2 show that the enzyme preparation splits ~-D-glucosides including was 5.8 when either buffer system was used. trehalose and starch, and also a fl-o-glucoside (cellobiose), but not a fl-o-galactoside (lactose) or the trisaccharide raffinose. Nature of ~-o-glucosidase
®->
® 7
1 .=
6 5
u E
E
=4
z3
v
"3
I=-
o=
2 1 J
..... 0
/
L
/
\
i
i
i
i
i
1
2
3
4
5
100
%
(cm) Fig. 3. Polyacrylamidegel electrophoresisof the fraction I of the affinity column, protein (Coomassie stain); . . . . . :tn-glucosidase activity• I.B. 9 / 2 - - F
L ......... Jo
50 Fraction No. ( 1 roll f r . )
Fig. 4. Isoelectricfocusing of the fraction I in a pH gradient 2.5-4 after 18-hrrun. Anode solution, 0.2 M phosphoric acid; cathode solution, 0.25 M NaOH. H Enzyme activity, O----O pH gradient.
f/
208
CHIHIRO KATAGIRI
10C
50
1
"5 rr o, o
0
2'0
4'0 5'0
60 70 80
Temperature ( ° C )
/ 0
Fig. 7. Effect of temperature on zt-D-glucosidase. The enzyme was incubated at various temperatures for 5 rain, and immediately placed at 30°C before assay of enzyme activity. Each point is the mean + S.E. of four experiments.
IIS ( mM -1 )
Fig. 5. Lineweaver-Burk plots of purified :~-D-glucosidase with various concentrations of PNPG.
DISCUSSION Polyacrylamide gel electrophoresis (Fig. 3) of fraction I shows the high homogeneity of the enzyme preparation and, since fraction I can hydrolyze melezitose but not act on raffinose, the presence of an ~-D-glucosidase in this fraction is evident. However, its substrate specificity suggested that it was contaminated with glycosidases other than s-Oglucosidase, for example, with trehalase (E.C.3.2.1.28), fl-D-glucosidase (E.C.3.2.1.21) and 7amylase (E.C.3.2.1.1). This contamination is to be expected as the adsorbent used for affinity
100
l\
/
v
chromatography contains a fl-glucosidic residue as a ligand and this is known not to be specific for ~-oglucosidase (MEGA and MATSUSHIMA, 1977). To eliminate such possible contaminants, further gel filtration was attempted, using Sephadex G-100 or Bio-Gel P-200. However, s~-o-glucosidase was always accompanied by other glycosidases, and appeared with the void volume of these columns. This may be explained by a possible formation o f a large complex consisti,ag of ~-D-glucosidase and other contaminants during the passage through such gels. Evidence has been obtained that :~-D-glucosidase exists in the serum of the American cockroach (KATAGIRI, 1979). It is interesting to compare the roidgut 7-D-glucosidase with the serum 7-oglucosidase. The K,, values are 5.0 and 2.8 mM, and p H optima 5.8 and 6.8, for the midgut enzyme and serum enzyme, respectively. The midgut enzyme is more heat-stable than the serum enzyme. These two enzymes exhibit different elution profiles on D E A E cellulose and affinity column chromatography, the serum enzyme is more easily eluted from the columns with lower concentration of the buffer. Also, the migration patterns of the two enzymes on polyacrylamide gels are different; the midgut enzyme migrates much faster than that of the serum, indicating
._> .5 5O
Table 2. Substrate specificity of glycosidases of fraction I
.>_-,
Sugar
(Structure)
Treha|ose Maltose Sucrose Turanose Cellobiose Lactose Maltotriose Melezitose Ra.ffinose Soluble starch
(Glc71 --*~1Glc)* (Glc~ 1~4Glc) (Glc~ 1~fl2Fru) (Glc~ 1--*3Fru) (Glcfll ---,4Glc) (Galfll ~4Glc) (Glca 1---,4G1c71--*4Glc) (Glc~ 1~ 3Frufl2---,~ 1Glc) (Gal~ 1--*6Glca 1-+f12Fru)
~aJ =_ 4
,
5
6 pH
7
B
Fig. 6. Effect of pH on ~-D-glucosidase. The enzyme was incubated with PNPG at different pH values for 15 rain at 30°C. H Citrate buffer, O O phosphate buffer. • Each point is the mean ___ S.E: of four experiments.
-
Hydrolysis + + + + + + + +
* Gal---galactose; Glc---glucose; Fru --fructose; + positive negative.
~t-o-Glucosidase in cockroach midgut Table 3. ~-D-Glucosidase in haemolymph of different insects Insects Lepidoptera Philosamia cynthia Bombyx mori Galleria mellonella Coleoptera Tenebrio molitor Hymenoptera Trichiocampus populi Diptera Sarcophaga peregrina
Stage Pupa Larva Larva
Activity
R
m
Adult Larva Larva
+ Present: - absent. t h a t the midgut enzyme is more acidic. In fact, Fig. 4 clearly d e m o n s t r a t e s t h a t isoelectric point of the enzyme is very low, p H 3.54, which c a n explain the a p p e a r a n c e of enzyme b a n d near the front o n polyacrylamide gel (Fig. 3). All the a b o v e d a t a suggests t h a t the midgut enzyme a n d the serum enzyme are different enzymes. Acknowledgements--I wish to thank Professor H. CHINO for his helpful discussions and advice and Dr. R. G. H. DOWNER of the University of Waterloo for critically reading the manuscript. This study was supported in part by research grants from the Ministry of Education of Japan and from Toray Foundation.
209
REFERENCES
DAVISB. J. (1964) Disc electrophoresis--lI. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121, 404--427. HUBERR. E. (1975) The purification and study of a honey bee abdominal sucrase exhibiting unusual solubility and kinetic properties. Archs Biochem. Biophys. 168, 198-209. HUBER R. E. and LEFEBVREY. A. ( 1971 ) The purification and some properties of soluble trehalase and sucrase from Drosophila melanogaster. Can. J. Biochem. 49, I 155-1164. KATAGIRI C. (1977) Localization of trehalase in the haemolymph of the American cockroach, Periplaneta americana. Insect Biochem 7, 351-353. KATAGIRI C. (1979) :t-D-Glucosidase in the serum of the American cockroach, Periplaneta americana. Insect Biochem. 9, 199-104. MARZLUF G. A. (1969) Studies of trehalase and sucrose of Drosophila melanogaster Archs Biochem Biophys, 134, 8-18. MEGA T. and MATSUSHIMA Y. (1977) Affinity chromatography of glycosidases--II. Studies on specific and non-specific binding. J. Biochem.81,571-578. SEMENZA G. (1976) Glucosidase of small intestinal brush border. In Membrane and Disease (Ed. by BOLLS L., HOFFMANN J. F. and LEAF A.), pp. 243-252. Ra',,~ii Fress, New York. WIGGLESWORTH V. B. (1927) CVI digestion in the cockroach--II. The digestion of carbohydrates. Biochem J. 21, 797-811. WILSON D. B. (1978) Cellular transport mechanisms. Ann Rev. Biochem. 47, 933-966.