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Inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 87 (2007) 73–77 www.elsevier.com/locate/ypest
Inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) a-glucosidase Jian-Fen Zhang, Yu-Guo Zheng *, Yin-Chu Shen Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China Received 9 April 2006; accepted 5 June 2006 Available online 10 June 2006
Abstract Valienamine, an aminocyclitol with similar configuration to a-glucose, has a strong inhibitory effect on a-glucosidase. a-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) a-glucosidase was investigated. Our results show that valienamine inhibition of honeybee a-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee aglucosidase in vitro with an IC50 value of 5.22 · 105 M and Ki value of 3.54 · 104 M at pH 6.5, 45 C. Valienamine has the potential to be developed into novel insecticides. 2006 Elsevier Inc. All rights reserved. Keywords: Insect a-glucosidase; Insecticide; Valienamine; Enzyme inhibition; Honeybee; Validamycins
1. Introduction Valienamine, [(1S,2S,3S,4R)-1-amino-5-(hydroxymethyl)-cyclohex-5-ene-2,3,4-triol], was first isolated from the microbial degradation of validamycin A by Pseudomonas denitrificans [1,2], and was later found in other organisms [3–5]. The degradation of validamycin A by Flavobacterium saccharophilum has been described. Validamycin A is first hydrolyzed to validoxylamine A by b-glucosidase. Then, validoxylamine A is degraded to validamine, valienamine and unsaturated ketocyclitols through 3-ketovalidoxylamine A by glucoside 3-dehydrogenase and 3-ketovalidoxylamine A C–N lyase [3,6]. Validamycin A was found to be a potent, reversible, and competitive inhibitor of Dictyostelium discoideum vegetative trehalase [7]. Validoxylamine A inhibits insect trehalase and prevents the use of trehalose as a source of energy for flight [8–10]. Valienamine has a similar configuration to a-D-glucose (Fig. 1), and has received considerable attention due to its strong inhibitory effect on mammalian and microbial *
Corresponding author. Fax: +86 571 88320630. E-mail address: [email protected] (Y.-G. Zheng).
0048-3575/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.06.001
a-glucosidase [11–14]. However, there have been few reports about the inhibitory effects of valienamine on insect a-glucosidase activity. a-Glucosidase (EC 3.2.1.20, a-glucoside glycohydrolase) is an exo-type carbohydrolase that catalyzes the liberation of a-glucoside from the nonreducing end of the substrate. This enzyme is widely distributed in microorganisms, plants, and animal tissues [15]. a-Glucosidase has a broad substrate specificity and is capable of hydrolyzing a wide range of a-D-glucosides including trehalose, maltose, and sucrose to glucose [16,17]. The honeybee (Apis cerana Fabr.) is a social insect, living in colonies composed of different castes, including a queen, workers, and drones. Three kinds of a-glucosidase, I, II, and III, were found from European honeybees Apis mellifera L. It was confirmed that a-glucosidase I was present in ventriculus, aglucosidase II was present in ventriculus and haemolymph, and a-glucosidase III was present in hypopharyngeal gland [18,19]. In the forager bee (older worker), the hypopharyngeal gland hydrolyzes sucrose present in nectar, to glucose and fructose by a-glucosidase [20,21]. The primary structure of a-glucosidase from hypopharyngeal gland of honeybee was closely related to those of fruit fly and mosquito
74
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
CH2OH
CH2OH
o OH
OH HO
NH2 OH
HO
OH OH
Valienamine Fig. 1. Chemical structure of valienamine and a-D-glucose.
maltase gene products [22]. Moreover, whiteflies, aphids, and other phloem-feeding insects ingested a diet containing nearly molar amounts of soluble carbohydrate, and hydrolyzed sucrose to glucose and fructose [23]. Thus in insects, a-glucosidase has a central role in carbohydrate metabolism. Glucosidase from insects has been shown to be inhibited by a number of compounds [15,16,22,23]. Glucosidase inhibitors can be important tools for study of their mechanisms of action, and might assist in the development of potent insecticides. Since the honeybee was an easy model to study, and the a-glucosidase from hypopharyngeal gland of honeybee was considered to be representative of other insects, we prepared partially purified a-glucosidase from hypopharyngeal gland of honeybee, and described the inhibitory characteristics and kinetics of valienamine against this a-glucosidase. 2. Materials and methods 2.1. Materials Honeybees were provided by Hangzhou Changqing Honey bees Co. Ltd. in Zhejiang, China. PNPG (p-nitrophenyl a-glucoside) and Econo-Pac High Q Cartridge anion exchange column (Bio-Rad, 5 ml) were obtained by their respective manufacturers. Valienamine was prepared according to method described previously [24] and was verified by NMR. All other chemicals were purchased from local suppliers and were of analytical grade. 2.2. Partially purified a-glucosidase preparation Crude a-glucosidase extract was prepared according to previously described method [25]. Sixty forager honeybees were killed by freezing at 20 C. Each frozen honeybee was cut to remove the body, and the heads were mashed with a glass rod in the presence of quartz sand in a glass bowl containing phosphate buffer (0.05 M, pH 7.5) for 30 min. After extraction at 4 C for 30 min, the mixture was centrifuged for 20 min at 12,000 rpm, and the supernatant solution was filtered through gauze to remove suspended particulates. The total volume of extract was 50 ml. Solid ammonium sulfate was added to the crude extract to 50% saturation. The resulting precipitate was removed by centrifugation at 12,000 rpm for 20 min. The
supernatant solution was brought to 80% saturation by further addition of ammonium sulfate. The resulting precipitate was collected by centrifugation at 12,000 rpm for 20 min and dissolved in 21 ml of phosphate buffer (0.05 M, pH 7.5). This solution was dialyzed against 20 mM phosphate buffer (pH 7.5) overnight. The dialyzed solution was added to an anion exchange column, Econo-Pac High Q Cartridge (Bio-Rad, 5 ml) employing a mobile phase of 0.05 M phosphate buffer (pH 7.5) at a flow rate of 1.0 ml/min. The absorbed proteins were eluted from the column with a linear gradient of NaCl from 0.05 to 0.5 M. Fractions having a-glucosidase activity were collected and served as the source of a-glucosidase. The enzyme was partially purified with the specific activity of 0.47 U/mg protein. 2.3. Assays of enzyme and protein The reaction mixture, containing 0.6 ml of 0.1 M phosphate buffer (pH 6.5), 0.1 ml of enzyme solution (about 0.2 U/ml) was preheated in a test tube using a water bath (45 C) for 10 min. The reaction was initiated by adding 0.3 ml of 20 mM a-PNPG (p-nitrophenyl a-glucoside) and incubated at 45 C for 10 min. The reaction was terminated by adding 4 ml of 0.1 M Na2CO3. Enzyme activity was quantified by measuring the p-nitrophenol released from a-PNPG at 400 nm. One unit of a-glucosidase activity was defined as the amount of enzyme required to produce 1 lM of p-nitrophenol per minute under the assay condition specified. The protein concentration was determined using the Bradford method [26]. A standard curve was established using bovine serum albumin as standard protein. 2.4. Enzyme inhibition assay The reaction mixture, containing 0.5 ml of 0.1 M phosphate buffer (pH 6.5), 0.1 ml of enzyme solution (about 0.2 U/ml), and 0.1 ml of valienamine (at several different concentration), was preheated at 45 C in a test tube for 10 min. Then, the residual enzyme activity was calculated using the following equation: Inhibition percentage ð%Þ ¼ ½ðUa Ue Þ=Ua 100% where Ua represents the a-glucosidase activity; Ue represents the a-glucosidase activity in the presence of valienamine. 3. Results and discussion 3.1. Effect of pH on inhibition of a-glucosidase by valienamine In order to verify the effect of valienamine on a-glucosidase activity at various pH value, a-glucosidase activity was examined using 50 mM sodium phosphate–citric acid buffer (pH 4–8) and 50 mM glycine–sodium hydroxide
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
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0 5
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9
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3.2. Effect of temperature on inhibition of a-glucosidase by valienamine In order to verify the effect of valienamine on a-glucosidase activity at various temperature, a-glucosidase activity was examined with 50 mM sodium phosphate–citric acid buffer (pH 6.5), at different temperatures (25–60 C) in the absence or presence of 5 · 105 M valienamine, respectively. As shown in Fig. 3, honeybee a-glucosidase activity was sensitive to temperature. The optimal temperature was 45 C both in the absence of or presence of valienamine. The shapes of these two curves were similar, and the
40
45
50
55
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0 65
Fig. 3. Effects of temperature on honeybee a-glucosidase activity in the absence (j) or presence of valienamine (d 5 · 105 M). The inhibition percentage (m) of a-glucosidase was almost at the same level.
inhibition percentage of a-glucosidase was almost at the same level at each temperature. This result suggested that the temperature did not affect the inhibition of valienamine on honeybee a-glucosidase. That was, the reaction of valienamine and honeybee a-glucosidase was not affected by temperature in this range. 3.3. Effect of valienamine concentration on inhibition The effect of valienamine concentration on inhibition of honeybee a-glucosidase was examined at the usual condition, pH 7.0, 37 C and at the optimal condition, pH 6.5, 45 C, respectively. As shown in Fig. 4, the concentration of valienamine required for 50% inhibition (IC50) of a-glucosidase was calculated to be 4.83 · 105 M at pH 7.0, 37 C and 5.22 · 105 M at pH 6.5, 45 C, respectively. That might be the reaction of valienamine and honeybee a-glucosidase was affected by pH value. This IC50 value was different from that of valienamine on other sources of a-glucosidase [12,24]. The possible reason might be that 100
Inhibition percent %
buffer (pH 8.5–10) in the absence or presence of 5 · 104 M valienamine, respectively. As shown in Fig. 2, the pH/activity curves obtained show narrow pH optima, in which the optimal pH was 6.5 in the absence of valienamine. However, the optimal pH of a-glucosidase from hypopharyngeal gland of honeybee (Apis mellifera L.) was 5.5 [18]. When valienamine was preincubated with a-glucosidase at 37 C for 10 min, the shapes of these two curves were similar, and the optimal pH was changed to 6.0. Thus, the presence of valienamine affected the optimal pH of a-glucosidase from honeybee slightly. The inhibition percentage of a-glucosidase was calculated using the same data. As showed in Fig. 2, the inhibition percentage varied at various pH values, and the minimal inhibition of valienamine was 54.5% at pH 6.0, which was the optimal pH of a-glucosidase in the presence of valienamine. That was, the reaction of valienamine and honeybee a-glucosidase was affected by pH value. However, the pH did not affect the inhibition of valienamine on the porcine small intestinal sucrase. The maximum activity was observed at pH 6.6 in the absence or presence of valienamine, and the same level of inhibition by valienamine was observed [24]. Probably, the effect of valienamine on a-glucosidase activity at various pH values was different when the source of the enzyme was different.
35
Temperature (centigrade)
pH value Fig. 2. Effects of pH value on honeybee a-glucosidase activity in the absence (j) or presence of valienamine (d 5 · 104 M). And the inhibition percentage (m) of a-glucosidase was varied from this pH range.
30
Inhibition percent (%)
60
Relative activity (%)
60
Inhibition percent %
Relative activity %
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0 0
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Concentration of valienamine 10 M Fig. 4. Effect of valienamine concentration on the activity of honeybee aglucosidase at (j) pH 7.0, 37 C and at (m) pH 6.5, 45 C, respectively.
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77 0.25
0.20 -1
the characteristic of a-glucosidase varies with the enzyme source. The curve of valienamine concentration vs. inhibition percentage was approximately linear over the concentration range from 105 to 104 M. This result indicated that the activity of a-glucosidase was reduced by valienamine in dose-dependent manner. Interestingly, the percentage of inhibition increased slowly when the concentration of valienamine reached 104 M. When the concentration of valienamine was more than 103 M, honeybee a-glucosidase was completely inhibited by valienamine.
1/V (mM/min)
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3.4. Effect of preincubation time on inhibition
0
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-1
1/S mM
In order to investigate whether the inhibitory effects of valienamine occur in a time-dependent manner, valienamine was added at different concentration of 5.0 · 104 M, 2.5 · 104 M, 1.0 · 104 M, 5.0 · 105 M, and 2.5 · 105 M, respectively, and the reactions were preincubated for different times (0–30 min). The inhibition curves of honeybee a-glucosidase by different concentration of valienamine over time were shown in Fig. 5. These results suggested that the action of valienamine on a-glucosidase was very rapid. Even when incubation time was zero, a-glucosidase inhibition was high. Thus, valienamine displayed no time-dependent inhibition of honeybee a-glucosidase. 3.5. Reversibility analysis of inhibition To determine whether the inhibition of a-glucosidase with valienamine was reversible, 1 ml of valienamine (5 · 103 M) was incubated with 0.5 ml of a-glucosidase (about 0.4 U/ml) for 1 h at 37 C. As a control, 1 ml of distilled water and 0.5 ml of the same preparation of a-glucosidase was incubated for 1 h at 37 C. Both reactions were dialyzed against phosphate buffer (20 mM, pH 7.0) at 4 C for 24 h, exchanging the buffer with fresh for four times. 100
Inhibition percent %
80
60
40
20
0 0
10
20
30
Time min Fig. 5. Effect of valienamine/enzyme preincubated time on the activity of honeybee a-glucosidase. The concentration of valienamine was 5.0 · 104 M. (j), 2.5 · 104 M (d), 1.0 · 104 M (m), 5.0 · 105 M (), and 2.5 · 105 M (w).
Fig. 6. Lineweaver-Burk double-reciprocal plots of the inhibition kinetic of honeybee a-glucosidase by valienamine. (j) The concentration of valienamine was 2.5 · 104 M; () control: no inhibitor was added.
An identical set of reactions was prepared and incubated at 4 C for 24 h without dialysis. All four reactions were diluted to determine the enzyme activity. The activity of a-glucosidase with dialysis was similar to that without dialysis. The activity of a-glucosidase incubated with valienamine was low as expected. a-Glucosidase recovered its original activity when valienamine was removed after dialysis. These results indicated that the inhibition of a-glucosidase by valienamine was reversible. However, the inhibition reversibility of a-glucosidase from other sources with valienamine was not detected [24]. 3.6. Kinetic analysis of inhibition Double-reciprocal plots of kinetic of inhibition on a-glucosidase with valienamine were shown in Fig. 6. The value of 1/V was a constant when valienamine was added, but Km value was increased. These results suggested that the inhibition of valienamine on a-glucosidase was competitive. The Km value of honeybee a-glucosidase was 2.55 · 103 M. The Ki value for valienamine was 3.54 · 104 M, which was about 10 times less than the Km value. However, in the experiment about inhibition of valienamine on porcine small intestinal sucrase, it had been found that the Ki value for valienamine was 7.7 · 104 M, which was about 100 times less than the Km value (5.0 · 102 M) of porcine small intestinal sucrase [24]. The date suggested that valienamine had greater affinity for active site of honeybee a-glucosidase than its substrate. Though the enzyme used in this study was not purified to be a homogeneous protein, the enzyme was partially purified and diluted thoroughly to remove low molecular chemicals. Therefore, inhibition of valienamine based on IC50 value and Ki value on a-glucosidase from honeybee hypopharyngeal gland was credible. However, the mechanism of inhibition of valienamine has not been studied at the cellular or molecular level, and further in vivo studies are needed. The information presented in this study about
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
the inhibitory effects of valienamine on honeybee a-glucosidase may provide useful information for the design of new inhibitors for insect a-glucosidase. Acknowledgments This work was supported by the National Fund of the Major Basic Research Development Program 973 of China (2003CB114402), the National Natural Science Foundation of China (20176055), and the Natural Science Foundation of Zhejiang Province (ZB0106). References [1] Y. Kameda, S. Horii, The unsaturated cyclitol part of the new antibiotics, the validamycins, J. Chem. Soc. Chem. Commun. 12 (1972) 746–747. [2] Y. Kameda, S. Horii, T. Yamano, Microbial transformation of validamycins, J. Antibiot. 28 (1975) 298–306. [3] N. Asano, M. Takeuchi, K. Ninomiya, Y. Kameda, K. Matsui, Microbial degradation of validamycin A by Flavobacterium saccharophilum, enzymatic cleavage of C–N linkage in validoxylamine A, J. Antibiot. 37 (8) (1984) 859–867. [4] Y.G. Zheng, X.F. Zhang, Y.C. Shen, Microbial transformation of validamycin A to valienamine by immobilized cells, Biocatal. Biotransfor. 23 (2005) 71–77. [5] Y.G. Zheng, Y.P. Xue, Y.C. Shen, Production of valienamine by a newly isolated strain: Stenotrophomonas maltrophilia, Enzyme Microb. Technol. (2006), in press. [6] J.F. Zhang, Y.G. Zheng, Y.P. Xue, Y.C. Shen, Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024, Appl. Microbiol. Biotechnol. (2006), in press. [7] T.A. Temesvari, D.A. Cotter, Trehalase of Dictyostelium discoideum: inhibition by amino-containing analogs of trehalose and affinity purification, Biochimie 79 (1997) 229–239. [8] Y.G. Zheng, L.Q. Jin, Y.C. Shen, Resin-catalyzed degradation of validamycin A for production of validoxylamine A, Catal. Commun. 5 (2004) 519–525. [9] Y.P. Xue, Y.G. Zheng, Y.C. Shen, Preparation of the trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validamycin A with honey-bee (Apis cerana Fabr.) b-glucosidase, Appl. Biochem. Biotechnol. 127 (2005) 157–171. [10] L.Q. Jin, Y.P. Xue, Y.G. Zheng, Y.C. Shen, Production of trehalase inhibitor validoxylamine A using acid-catalyzed hydrolysis of validamycin A, Catal. Commun. 7 (2006) 157–161. [11] X.L. Chen, Y.G. Zheng, Y.C. Shen, Voglibose (Basen,AO-128), one of the most important a-glucosidase inhibitor, Curr. Med. Chem. 13 (2006) 109–116.
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[12] Y. Kameda, N. Asano, M. Yoshikawa, K. Matsui, Valienamine as an a-glucosidase inhibition, J. Antibiot. 33 (1980) 1575–1576. [13] Y. Kameda, N. Asano, M. Yoshikawa, M. Takeuchi, T. Yamaguchi, K. Matsui, Valiolamine, a new a-glucosidase inhibiting aminocyclitol produced by Streptomyces hygroscopicus, J. Antibiot. 37 (1984) 1301–1307. [14] X.L. Chen, Y.X. Fan, Y.G. Zheng, Y.C. Shen, Properties and production of valienamine and its related analogues, Chem. Rev. 103 (2003) 1955–1977. [15] A. Kimura, J.H. Lee, I.S. Lee, H.S. Lee, K.H. Park, S. Chiba, D. Kim, Two potent competitive inhibitors discriminating a-glucosidase family I, from family II, Carbohydr. Res. 339 (2004) 1035–1040. [16] A.M. Scofield, P. Witham, R.J. Nash, G.C. Kite, L.E. Fellows, Castanospermine and other polyhydroxy alkaloids as inhibitors of insect glycosidase, Comp. Biochem. Physiol. 112A (1995) 187–196. [17] Y. Xia, J.M. Clarkson, A.K. Charnley, Trehalose-hydrolysing enzymes of Metarhiziun anisopliae and their role in pathogenesis of the tobacco hornworm, Manduca Sexta, J. Invertebr. pathol. 80 (2002) 139–147. [18] M. Nishimoto, M. Kubota, M. Tsuji, H. Mori, A. Kimura, H. Matsui, S. Chiba, Purification and substrate specificity of honeybee, Apis mellifera L., a-glucosidase III, Biosci. Biotechnol. Biochem. 65 (7) (2001) 1610–1616. [19] M. Kubota, M. Tsuji, M. Nishimoto, J. Wongchawalit, M. Okuyama, H. Mori, H. Matsui, Localization of a-glucosidase I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of aglucosidase in honey, Biosci. Biotechnol. Biochem. 68 (11) (2004) 2346–2352. [20] K.S. Santos, L.D. Santos, M.A. Mendes, B.M. Souza, O. Malaspina, M.S. Palma, Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.), Insect Biochem. Mol. 35 (2005) 85–91. [21] K. Ohashi, M. Sawata, H. Takeuchi, S. Natori, T. Kubo, Molecular cloning of cDNA and analysis of expression of the gene for a-glucosidase from the hypopharyngeal gland of honeybee Apis mellifera L., Biochem. Biophy. Res. Commun. 221 (1996) 380–385. [22] M.E. Salvucci, Effect of the a-glucosidase inhibitor, bromoconduritol, on carbohydrate metabolism in the silverleaf whitefly, Bemisia argentifolii, Arch. Insect Biochem. 45 (2000) 117–128. [23] E.S.H.El. Ashry, N. Rashed, A.H.S. Shobier, Glycosidase inhibitors and their chemotherapeutic value, part 2, Pharmazie 55 (2) (2000) 331–348. [24] Y.G. Zheng, X.P. Shentu, Y.C. Shen, Inhibition of porcine small intestinal sucrase by valienamine, J. Enzyme Inhib. Med. Chem. 20 (2005) 49–53. [25] J. Pontoh, N.H. Low, Purification and characterization of bglucosidase from honey bees (Apis mellifera), Insect Biochem. Mol. 32 (2002) 679–690. [26] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254.
Pesticide Biochemistry and Physiology 87 (2007) 73–77 www.elsevier.com/locate/ypest
Inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) a-glucosidase Jian-Fen Zhang, Yu-Guo Zheng *, Yin-Chu Shen Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China Received 9 April 2006; accepted 5 June 2006 Available online 10 June 2006
Abstract Valienamine, an aminocyclitol with similar configuration to a-glucose, has a strong inhibitory effect on a-glucosidase. a-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) a-glucosidase was investigated. Our results show that valienamine inhibition of honeybee a-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee aglucosidase in vitro with an IC50 value of 5.22 · 105 M and Ki value of 3.54 · 104 M at pH 6.5, 45 C. Valienamine has the potential to be developed into novel insecticides. 2006 Elsevier Inc. All rights reserved. Keywords: Insect a-glucosidase; Insecticide; Valienamine; Enzyme inhibition; Honeybee; Validamycins
1. Introduction Valienamine, [(1S,2S,3S,4R)-1-amino-5-(hydroxymethyl)-cyclohex-5-ene-2,3,4-triol], was first isolated from the microbial degradation of validamycin A by Pseudomonas denitrificans [1,2], and was later found in other organisms [3–5]. The degradation of validamycin A by Flavobacterium saccharophilum has been described. Validamycin A is first hydrolyzed to validoxylamine A by b-glucosidase. Then, validoxylamine A is degraded to validamine, valienamine and unsaturated ketocyclitols through 3-ketovalidoxylamine A by glucoside 3-dehydrogenase and 3-ketovalidoxylamine A C–N lyase [3,6]. Validamycin A was found to be a potent, reversible, and competitive inhibitor of Dictyostelium discoideum vegetative trehalase [7]. Validoxylamine A inhibits insect trehalase and prevents the use of trehalose as a source of energy for flight [8–10]. Valienamine has a similar configuration to a-D-glucose (Fig. 1), and has received considerable attention due to its strong inhibitory effect on mammalian and microbial *
Corresponding author. Fax: +86 571 88320630. E-mail address: [email protected] (Y.-G. Zheng).
0048-3575/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.06.001
a-glucosidase [11–14]. However, there have been few reports about the inhibitory effects of valienamine on insect a-glucosidase activity. a-Glucosidase (EC 3.2.1.20, a-glucoside glycohydrolase) is an exo-type carbohydrolase that catalyzes the liberation of a-glucoside from the nonreducing end of the substrate. This enzyme is widely distributed in microorganisms, plants, and animal tissues [15]. a-Glucosidase has a broad substrate specificity and is capable of hydrolyzing a wide range of a-D-glucosides including trehalose, maltose, and sucrose to glucose [16,17]. The honeybee (Apis cerana Fabr.) is a social insect, living in colonies composed of different castes, including a queen, workers, and drones. Three kinds of a-glucosidase, I, II, and III, were found from European honeybees Apis mellifera L. It was confirmed that a-glucosidase I was present in ventriculus, aglucosidase II was present in ventriculus and haemolymph, and a-glucosidase III was present in hypopharyngeal gland [18,19]. In the forager bee (older worker), the hypopharyngeal gland hydrolyzes sucrose present in nectar, to glucose and fructose by a-glucosidase [20,21]. The primary structure of a-glucosidase from hypopharyngeal gland of honeybee was closely related to those of fruit fly and mosquito
74
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
CH2OH
CH2OH
o OH
OH HO
NH2 OH
HO
OH OH
Valienamine Fig. 1. Chemical structure of valienamine and a-D-glucose.
maltase gene products [22]. Moreover, whiteflies, aphids, and other phloem-feeding insects ingested a diet containing nearly molar amounts of soluble carbohydrate, and hydrolyzed sucrose to glucose and fructose [23]. Thus in insects, a-glucosidase has a central role in carbohydrate metabolism. Glucosidase from insects has been shown to be inhibited by a number of compounds [15,16,22,23]. Glucosidase inhibitors can be important tools for study of their mechanisms of action, and might assist in the development of potent insecticides. Since the honeybee was an easy model to study, and the a-glucosidase from hypopharyngeal gland of honeybee was considered to be representative of other insects, we prepared partially purified a-glucosidase from hypopharyngeal gland of honeybee, and described the inhibitory characteristics and kinetics of valienamine against this a-glucosidase. 2. Materials and methods 2.1. Materials Honeybees were provided by Hangzhou Changqing Honey bees Co. Ltd. in Zhejiang, China. PNPG (p-nitrophenyl a-glucoside) and Econo-Pac High Q Cartridge anion exchange column (Bio-Rad, 5 ml) were obtained by their respective manufacturers. Valienamine was prepared according to method described previously [24] and was verified by NMR. All other chemicals were purchased from local suppliers and were of analytical grade. 2.2. Partially purified a-glucosidase preparation Crude a-glucosidase extract was prepared according to previously described method [25]. Sixty forager honeybees were killed by freezing at 20 C. Each frozen honeybee was cut to remove the body, and the heads were mashed with a glass rod in the presence of quartz sand in a glass bowl containing phosphate buffer (0.05 M, pH 7.5) for 30 min. After extraction at 4 C for 30 min, the mixture was centrifuged for 20 min at 12,000 rpm, and the supernatant solution was filtered through gauze to remove suspended particulates. The total volume of extract was 50 ml. Solid ammonium sulfate was added to the crude extract to 50% saturation. The resulting precipitate was removed by centrifugation at 12,000 rpm for 20 min. The
supernatant solution was brought to 80% saturation by further addition of ammonium sulfate. The resulting precipitate was collected by centrifugation at 12,000 rpm for 20 min and dissolved in 21 ml of phosphate buffer (0.05 M, pH 7.5). This solution was dialyzed against 20 mM phosphate buffer (pH 7.5) overnight. The dialyzed solution was added to an anion exchange column, Econo-Pac High Q Cartridge (Bio-Rad, 5 ml) employing a mobile phase of 0.05 M phosphate buffer (pH 7.5) at a flow rate of 1.0 ml/min. The absorbed proteins were eluted from the column with a linear gradient of NaCl from 0.05 to 0.5 M. Fractions having a-glucosidase activity were collected and served as the source of a-glucosidase. The enzyme was partially purified with the specific activity of 0.47 U/mg protein. 2.3. Assays of enzyme and protein The reaction mixture, containing 0.6 ml of 0.1 M phosphate buffer (pH 6.5), 0.1 ml of enzyme solution (about 0.2 U/ml) was preheated in a test tube using a water bath (45 C) for 10 min. The reaction was initiated by adding 0.3 ml of 20 mM a-PNPG (p-nitrophenyl a-glucoside) and incubated at 45 C for 10 min. The reaction was terminated by adding 4 ml of 0.1 M Na2CO3. Enzyme activity was quantified by measuring the p-nitrophenol released from a-PNPG at 400 nm. One unit of a-glucosidase activity was defined as the amount of enzyme required to produce 1 lM of p-nitrophenol per minute under the assay condition specified. The protein concentration was determined using the Bradford method [26]. A standard curve was established using bovine serum albumin as standard protein. 2.4. Enzyme inhibition assay The reaction mixture, containing 0.5 ml of 0.1 M phosphate buffer (pH 6.5), 0.1 ml of enzyme solution (about 0.2 U/ml), and 0.1 ml of valienamine (at several different concentration), was preheated at 45 C in a test tube for 10 min. Then, the residual enzyme activity was calculated using the following equation: Inhibition percentage ð%Þ ¼ ½ðUa Ue Þ=Ua 100% where Ua represents the a-glucosidase activity; Ue represents the a-glucosidase activity in the presence of valienamine. 3. Results and discussion 3.1. Effect of pH on inhibition of a-glucosidase by valienamine In order to verify the effect of valienamine on a-glucosidase activity at various pH value, a-glucosidase activity was examined using 50 mM sodium phosphate–citric acid buffer (pH 4–8) and 50 mM glycine–sodium hydroxide
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
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3.2. Effect of temperature on inhibition of a-glucosidase by valienamine In order to verify the effect of valienamine on a-glucosidase activity at various temperature, a-glucosidase activity was examined with 50 mM sodium phosphate–citric acid buffer (pH 6.5), at different temperatures (25–60 C) in the absence or presence of 5 · 105 M valienamine, respectively. As shown in Fig. 3, honeybee a-glucosidase activity was sensitive to temperature. The optimal temperature was 45 C both in the absence of or presence of valienamine. The shapes of these two curves were similar, and the
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50
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Fig. 3. Effects of temperature on honeybee a-glucosidase activity in the absence (j) or presence of valienamine (d 5 · 105 M). The inhibition percentage (m) of a-glucosidase was almost at the same level.
inhibition percentage of a-glucosidase was almost at the same level at each temperature. This result suggested that the temperature did not affect the inhibition of valienamine on honeybee a-glucosidase. That was, the reaction of valienamine and honeybee a-glucosidase was not affected by temperature in this range. 3.3. Effect of valienamine concentration on inhibition The effect of valienamine concentration on inhibition of honeybee a-glucosidase was examined at the usual condition, pH 7.0, 37 C and at the optimal condition, pH 6.5, 45 C, respectively. As shown in Fig. 4, the concentration of valienamine required for 50% inhibition (IC50) of a-glucosidase was calculated to be 4.83 · 105 M at pH 7.0, 37 C and 5.22 · 105 M at pH 6.5, 45 C, respectively. That might be the reaction of valienamine and honeybee a-glucosidase was affected by pH value. This IC50 value was different from that of valienamine on other sources of a-glucosidase [12,24]. The possible reason might be that 100
Inhibition percent %
buffer (pH 8.5–10) in the absence or presence of 5 · 104 M valienamine, respectively. As shown in Fig. 2, the pH/activity curves obtained show narrow pH optima, in which the optimal pH was 6.5 in the absence of valienamine. However, the optimal pH of a-glucosidase from hypopharyngeal gland of honeybee (Apis mellifera L.) was 5.5 [18]. When valienamine was preincubated with a-glucosidase at 37 C for 10 min, the shapes of these two curves were similar, and the optimal pH was changed to 6.0. Thus, the presence of valienamine affected the optimal pH of a-glucosidase from honeybee slightly. The inhibition percentage of a-glucosidase was calculated using the same data. As showed in Fig. 2, the inhibition percentage varied at various pH values, and the minimal inhibition of valienamine was 54.5% at pH 6.0, which was the optimal pH of a-glucosidase in the presence of valienamine. That was, the reaction of valienamine and honeybee a-glucosidase was affected by pH value. However, the pH did not affect the inhibition of valienamine on the porcine small intestinal sucrase. The maximum activity was observed at pH 6.6 in the absence or presence of valienamine, and the same level of inhibition by valienamine was observed [24]. Probably, the effect of valienamine on a-glucosidase activity at various pH values was different when the source of the enzyme was different.
35
Temperature (centigrade)
pH value Fig. 2. Effects of pH value on honeybee a-glucosidase activity in the absence (j) or presence of valienamine (d 5 · 104 M). And the inhibition percentage (m) of a-glucosidase was varied from this pH range.
30
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Inhibition percent %
Relative activity %
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Concentration of valienamine 10 M Fig. 4. Effect of valienamine concentration on the activity of honeybee aglucosidase at (j) pH 7.0, 37 C and at (m) pH 6.5, 45 C, respectively.
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77 0.25
0.20 -1
the characteristic of a-glucosidase varies with the enzyme source. The curve of valienamine concentration vs. inhibition percentage was approximately linear over the concentration range from 105 to 104 M. This result indicated that the activity of a-glucosidase was reduced by valienamine in dose-dependent manner. Interestingly, the percentage of inhibition increased slowly when the concentration of valienamine reached 104 M. When the concentration of valienamine was more than 103 M, honeybee a-glucosidase was completely inhibited by valienamine.
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3.4. Effect of preincubation time on inhibition
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In order to investigate whether the inhibitory effects of valienamine occur in a time-dependent manner, valienamine was added at different concentration of 5.0 · 104 M, 2.5 · 104 M, 1.0 · 104 M, 5.0 · 105 M, and 2.5 · 105 M, respectively, and the reactions were preincubated for different times (0–30 min). The inhibition curves of honeybee a-glucosidase by different concentration of valienamine over time were shown in Fig. 5. These results suggested that the action of valienamine on a-glucosidase was very rapid. Even when incubation time was zero, a-glucosidase inhibition was high. Thus, valienamine displayed no time-dependent inhibition of honeybee a-glucosidase. 3.5. Reversibility analysis of inhibition To determine whether the inhibition of a-glucosidase with valienamine was reversible, 1 ml of valienamine (5 · 103 M) was incubated with 0.5 ml of a-glucosidase (about 0.4 U/ml) for 1 h at 37 C. As a control, 1 ml of distilled water and 0.5 ml of the same preparation of a-glucosidase was incubated for 1 h at 37 C. Both reactions were dialyzed against phosphate buffer (20 mM, pH 7.0) at 4 C for 24 h, exchanging the buffer with fresh for four times. 100
Inhibition percent %
80
60
40
20
0 0
10
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30
Time min Fig. 5. Effect of valienamine/enzyme preincubated time on the activity of honeybee a-glucosidase. The concentration of valienamine was 5.0 · 104 M. (j), 2.5 · 104 M (d), 1.0 · 104 M (m), 5.0 · 105 M (), and 2.5 · 105 M (w).
Fig. 6. Lineweaver-Burk double-reciprocal plots of the inhibition kinetic of honeybee a-glucosidase by valienamine. (j) The concentration of valienamine was 2.5 · 104 M; () control: no inhibitor was added.
An identical set of reactions was prepared and incubated at 4 C for 24 h without dialysis. All four reactions were diluted to determine the enzyme activity. The activity of a-glucosidase with dialysis was similar to that without dialysis. The activity of a-glucosidase incubated with valienamine was low as expected. a-Glucosidase recovered its original activity when valienamine was removed after dialysis. These results indicated that the inhibition of a-glucosidase by valienamine was reversible. However, the inhibition reversibility of a-glucosidase from other sources with valienamine was not detected [24]. 3.6. Kinetic analysis of inhibition Double-reciprocal plots of kinetic of inhibition on a-glucosidase with valienamine were shown in Fig. 6. The value of 1/V was a constant when valienamine was added, but Km value was increased. These results suggested that the inhibition of valienamine on a-glucosidase was competitive. The Km value of honeybee a-glucosidase was 2.55 · 103 M. The Ki value for valienamine was 3.54 · 104 M, which was about 10 times less than the Km value. However, in the experiment about inhibition of valienamine on porcine small intestinal sucrase, it had been found that the Ki value for valienamine was 7.7 · 104 M, which was about 100 times less than the Km value (5.0 · 102 M) of porcine small intestinal sucrase [24]. The date suggested that valienamine had greater affinity for active site of honeybee a-glucosidase than its substrate. Though the enzyme used in this study was not purified to be a homogeneous protein, the enzyme was partially purified and diluted thoroughly to remove low molecular chemicals. Therefore, inhibition of valienamine based on IC50 value and Ki value on a-glucosidase from honeybee hypopharyngeal gland was credible. However, the mechanism of inhibition of valienamine has not been studied at the cellular or molecular level, and further in vivo studies are needed. The information presented in this study about
J.-F. Zhang et al. / Pesticide Biochemistry and Physiology 87 (2007) 73–77
the inhibitory effects of valienamine on honeybee a-glucosidase may provide useful information for the design of new inhibitors for insect a-glucosidase. Acknowledgments This work was supported by the National Fund of the Major Basic Research Development Program 973 of China (2003CB114402), the National Natural Science Foundation of China (20176055), and the Natural Science Foundation of Zhejiang Province (ZB0106). References [1] Y. Kameda, S. Horii, The unsaturated cyclitol part of the new antibiotics, the validamycins, J. Chem. Soc. Chem. Commun. 12 (1972) 746–747. [2] Y. Kameda, S. Horii, T. Yamano, Microbial transformation of validamycins, J. Antibiot. 28 (1975) 298–306. [3] N. Asano, M. Takeuchi, K. Ninomiya, Y. Kameda, K. Matsui, Microbial degradation of validamycin A by Flavobacterium saccharophilum, enzymatic cleavage of C–N linkage in validoxylamine A, J. Antibiot. 37 (8) (1984) 859–867. [4] Y.G. Zheng, X.F. Zhang, Y.C. Shen, Microbial transformation of validamycin A to valienamine by immobilized cells, Biocatal. Biotransfor. 23 (2005) 71–77. [5] Y.G. Zheng, Y.P. Xue, Y.C. Shen, Production of valienamine by a newly isolated strain: Stenotrophomonas maltrophilia, Enzyme Microb. Technol. (2006), in press. [6] J.F. Zhang, Y.G. Zheng, Y.P. Xue, Y.C. Shen, Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024, Appl. Microbiol. Biotechnol. (2006), in press. [7] T.A. Temesvari, D.A. Cotter, Trehalase of Dictyostelium discoideum: inhibition by amino-containing analogs of trehalose and affinity purification, Biochimie 79 (1997) 229–239. [8] Y.G. Zheng, L.Q. Jin, Y.C. Shen, Resin-catalyzed degradation of validamycin A for production of validoxylamine A, Catal. Commun. 5 (2004) 519–525. [9] Y.P. Xue, Y.G. Zheng, Y.C. Shen, Preparation of the trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validamycin A with honey-bee (Apis cerana Fabr.) b-glucosidase, Appl. Biochem. Biotechnol. 127 (2005) 157–171. [10] L.Q. Jin, Y.P. Xue, Y.G. Zheng, Y.C. Shen, Production of trehalase inhibitor validoxylamine A using acid-catalyzed hydrolysis of validamycin A, Catal. Commun. 7 (2006) 157–161. [11] X.L. Chen, Y.G. Zheng, Y.C. Shen, Voglibose (Basen,AO-128), one of the most important a-glucosidase inhibitor, Curr. Med. Chem. 13 (2006) 109–116.
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