Purification partial characterization and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 2:415-428 (1985) Purification, Partial Characterization, and Postembryonic Levels of Amylases From Sitophilus oryzae and Sitophilus granarius J.E. Baker and S.M. Woo Stored Product Insects Research and Development Laboratory, Agricultural Research Sewice, U.S.Department of Agricidture, Savannalz, Georgia Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were purified by using a sequential procedure of ammonium sulfate precipitation, glycogen-complex formation, and ion exchange chromatography. Amylase of S. oryaze was purified 47.4-fold to a specific activity of 478 unitdmg protein. One amylase unit equals 1 mg maltose hydrate producedlmin at 3OOC. Amylase of S. granarius was purified 85.4fold to a specific activity of 453 u n i t s h g protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a single protein with an isoelectric point of pH 3.76. Purified amylases of both species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had double energies of activation calculated from Arrhenius plots. Based o n fresh weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae contained three-fold and eight-fold more amylase than S. granarius and S. zeamais Motschulsky, respectively. High amylase levels in S. oryzae may provide this species with an adaptive advantage when feeding o n cereals containing naturally occurring amylase inhibitors. Key words: Sitophilus, S. oryzae, S. granarius, S. zeamais, rice weevil, granary weevil, maize weevil, amylase, purification, digestion, cereals, feeding, amylase inhibitors, adaptive significance INTRODUCTION Amylases from larvae of the granary weevil, Sitophilus granarius (L,),and the maize weevil, S. zeamais Motschulsky, are active in mildly acid buffers, Mention of a proprietary product does not constitute a recommendation by the U.S. Department of Agriculture. Received February 25,1985;accepted May 1,1985. Address reprint requests to J.E. Baker, USDA-ARS, P.O. Box 22909,Savannah, GA 31403. 0 1985 Alan R. Liss, Inc. Baker and Woo 416 are activated by C1-, and are stabilized against thermal inactivation by Ca2+ [l].The amylases are of the endoamylase type (E.C.126.96.36.199) and hydrolyze soluble starch and amylopectin with identical kinetic parameters. Electrophorectic analysis of larval midgut homogenates on polyacrylamide slab gels indicated that S. zeurnais had two strongly anionic amylase isozymes, while a single amylase was found in S. grunarius. Gel patterns of adult midgut homogenates indicated that larvae and adults of a given species had identical amylase isozymes . Evidence also indicated that, in contrast to proteinases that were secreted into the lumen by midgut epithelial cells, the origin of amylase in adult S. grunurius was the salivary glands . Adults of Sitophilus feeding on wheat live for extended periods and consume significantly more food than developing stages . Since starch makes up 55% of the wheat kernel and is the predominant nutrient in endosperm [S], the relatively high levels of amylase present in Sitophilus  are important in food utilization processes in these species. The role of amylase in starch digestion by the weevils is complicated by the presence of potent a-amylase inhibitors present in wheat . These inhibitors interact with amylases from Tenebrio rnolitor L. [6,8], Triboliurn custaneurn Herbst , as well as Sitophilus [Z6/10] To study the in vitro interactions of inhibitor with enzyme, purified amylases are required. A procedure based on ammonium sulfate precipitation, formation of an insoluble glycogen-amylase complex, and ion exchange chromatography on DEAE-SephaceP was developed and used to purify the amylases from adults of the rice weevil, S. oryzue (L.) and S. granarius. In addition, several properties of the purified amylases as well as postembryonic levels of amylase activity in S. oryzue, S. grunurius, and S. zeurnais were compared. 1 MATERIALS AND METHODS Insects Adults of S. oryzue and S. granurius were removed from cultures maintained on soft, red winter wheat at 27°C and 50-609'0 RH with a 12:12 LD photoperiod. Assays Amylase activity was measured with the DNS* procedure of Bernfeld [ll] slightly modified from that of Baker El]. Activity at each step in the purification sequence was determined by adding 5 pl of sample to 1 ml 1%soluble starch (potato, Lintner grade, Sigma Chemical Co., St. Louis, MO) in 20 mh4 acetate, pH 5.0, containing 20 mh4 NaCl and 0.1 mM CaC12. After 1 min at 30°C the reaction was stopped by adding 1 ml DNS, the tubes heated in 'Abbreviations: 3,5-dinitrosalicylic acid = DNS; energy of activation = Ea; molecular weight = MW; polyacrylarnide gel electrophoresis = PAGE; mobility relative to brornophenol blue = Rm; sodium dodecyl sulfate-polyacrylarnide gel electrophoresis = SDS-PAGE; tris(hydroxymethy1)aminomethane = tris. Purificationand Amylases 417 boiling water for 5 min, cooled, diluted with 2 ml H20, and read at 550 nm. Reaction times for amylase from the final purification step were reduced to 30 sec and in later tests enzyme samples were diluted to maintain linearity. Maltose was used as a standard. A unit of amylase activity was defined as the amount of enzyme that produced 1mg of maltose hydrate per minute at 30°C. Specific activity was defined as units per milligram of protein. Protein was estimated with the procedure of Lowry et a1 . Column fractions were monitored for amylase activity by measuring residual starch with a KI-iodine procedure . Purification Procedure Adults of S. oryuze (20.0 g) and S. grunarius (16.3 g) were ground in 1% NaCl (2 mllg) with a mortar and pestle, rinsed with 1%NaCl (1mllg) and the total slurry centrifuged at 5,300g for 40 min. The supernatants were decanted and recentrifuged to remove floating lipoidal material prior to ammonium sulfate precipitation. Ammonium sulfate was stirred in at 0°C to 1.5 M and the precipitate that formed was removed by centrifugation. Additional ammonium sulfate was added to 4.0 M and the heavy precipitate that formed after 60 min was collected by centrifugation for 40 min at 5,300g. The supernatant was discarded and the pellet dissolved in standard buffer (20 mM acetate, pH 5.0, containing 20 mM NaCl and 0.01 mM CaC12). Amylase was precipitated from the buffer solution by formation of an alcohol-insoluble glycogen-amylase complex by using the procedure of Loyter and Schramm . Because of the extreme activity of the amylases in these species, glycogen was added at the rate of 1 mgl5 units of amylase activity. The glycogen-amylase complex, insoluble in cold standard buffer containing 40% ethanol, was collected by centrifugation and washed with ethanolic buffer. Following centrifugation the pellet was dissolved in buffer and incubated for 3 h at 37°C to remove glycogen by hydrolysis. Hydrolytic products were separated from enzyme via ultrafiltration on YM-10 membranes on an Amicon stirred cell. When the ultrafiltrate no longer gave a positive DNS reaction, the standard acetate buffer was exchanged on the stirred cell for 20 mM tris-chloride, pH 7.5, containing 0.1 mM CaCl,. Following concentration, samples were applied to a DEAE-SephaceP (Pharmacia, Piscataway, NJ) column (1.6 x 13cm) equilibrated in 20 mh4 trischloride, pH 7.5, with 0.1 mM CaC12. Proteins were eluted with a linear gradient of 250 m10.4 M NaCl in the tris buffer into 250 ml tris buffer. Initial flow rate was 1 mllmin and 4-ml fractions were collected. Alternate tubes were checked for absorbance at 280 nm and for amylase activity by using the KI-iodine procedure for residual starch. Tubes containing amylase were combined and concentrated via ultrafiltration. Absorbance values of amylases in distilled H20 were determined at 280 nm in 1-cm pathlength cuvettes. Electrophoresis Purified amylases were examined by PAGE on 7.5% gels according to Davis  and Baker 1161. SDS-PAGE on 10% gels was performed according 418 Baker and Woo to LKB manual 2001 except SDS was omitted from the gels and the SDS concentration in the electrode buffer was reduced to 0.05%. PAGE and SDSPAGE gels were stained with Coomassie brilliant blue R-250. Protein standards were obtained from Sigma and BioRad (Richmond, CA). Plots of log molecular weight versus mobility relative to bromophenol blue were prepared. Isoelectric points of the amylases were determined by isoelectric focusing on polyacrylamide gels containing Pharmalyte 2.5-5.0 range ampholytes (Pharmacia) according to procedures previously described [lq, Kinetic Parameters Kinetic parameters for hydrolysis of starch were determined from duplicate anaylses of three protein concentrations of purified amylases from each species reacting with eight concentrations of starch from 0.066 to 0.5% in 50 mM sodium acetate, pH 5.0, containing 20 mM NaCl and 0.1 mM CaC12for 2 min at 30°C. Protein concentrations used were 0.3, 0.45, and 0.6 p g for S. o y z u e and 0.29, 0.39, and 0.48 p g for S. grunuuius. Each tube contained 1.0 ml starch solution. Double reciprocal plots of velocity against starch concentration were prepared. Mean intercept values were obtained from plots using a linear program analysis (Hewlett-Packard Model 97). Km (as percentage of starch in reaction mixture) and Vmax were estimated for each species. Effect of chloride on hydrolysis of starch was determined as above except purified enzymes were diluted and washed with sodium acetate buffer (without chloride) on Centricon 10 Microconcentrator@tubes prior to assay. Starch solutions were prepared in acetate buffer with and without 20 mM NaCl and 0.1 mM CaC12. Double reciprocal plots were prepared as above. Effect of Temperature on Amylase Activity Activity of purified amylases was determined at 5°C intervals from 10°C to 50°C. Mean values obtained from two tests of amylases of each species with triplicate analyses at each temperature were used to prepare Arrhenius plots. Ea values were calculated from the equation: Ea = -2.303R X (slope of 1I"K vs log v). Slopes of 1I"K ( X lo3)vs log v between given temperatures were determined from a linear analysis program. Comparative Levels of Amylase in Sitophilus Amylase levels in whole body extracts of larvae and adults of Sitophilus through 10 weeks of age were compared. Newly hatched larvae, 7-8-day-old larvae, 15-18-day-old larvae, pupae, and newly eclosed adults of S. oryzae and S. grunurius were obtained from groups reared on a casein-starch-based meridic diet [18,19]. Groups of newly emerged adults of S. oyzue and S. grunurius (from cultures reared on whole wheat) and S. zeumuis (from cultures reared on whole corn) were placed on fresh whole wheat in 3.7-liter jars for ad libitum feeding at 28°C and 50-60% RH in continuous darkness. At weekly intervals for 10 weeks, three groups of ten weevils of each species were removed, weighed, homogenized in 0.5 ml cold H20, the homogenates centrifuged in 1.5-ml tubes, and triplicate 2 0 4 aliquots of supernatant analyzed for amylase and protein. Amylase assays for S. oryzue were conducted Purificationand Amylases 419 for 1min at 30°C while assays for S. grunurius and S. zeurnuis were conducted for 2 min. Amylase activity was expressed as milliunits per milligram of protein or units per milligram fresh weight. Activity based on fresh weight was determined by calculating total units of activity on a per midgut basis and dividing by mean fresh weight of insects used in that particular replicate. Whole body extracts of larvae were prepared as above except only five mature larvae (15-18 days old) were used per replicate. Adults of S. zurnuis feeding on wheat were analyzed at biweekly intervals after four weeks of age. Weevils of all three species feeding on wheat were removed at 3-week intervals and placed on clean wheat to prevent mixing of newly emerged weevils with the original weevils of known age. RESULTS Purification of Amylases Results of purification procedures for amylase from adults of S. oryzue are presented in Table 1. Amylase complexed with glycogen on a nearly quantitative basis, resulting in a 12-fold purification over the ammonium sulfate step and a 20.8-fold purification relative to the crude homogenate. Amylase from S. oryme eluted from the DEAE-SephaceP column in a single, symetrical peak with 0.3 M NaCl in buffer at pH 7.5 (Fig. 1).Tubes 94 to 102 were combined and concentrated. Specific activity after column chromatography was 478.6 unitslmg protein, which represented a 47.4-fold increase over that of the original homogenate. Final recovery was 59% of original activity. Absorbance coefficient (Alyo, 280 nm) of the purified amylase of S. oqzue was 13.3. Amylase from S. gvunurius was isolated and purified by using the same sequence of procedures used for S. oryzue. The glycogen-complex step resulted in a 28-fold increase in specific activity over the ammonium sulfate step and a 45-fold increase in activity over the original homogenate (Table 2). A single, symetrical peak of amylase activity was eluted from the DEAESephaceP column with 0.32 M NaCl in buffer at pH 7.5. Tubes 104 to 110 were combined and concentrated. Although specific activity of the original homogenate of S. grunurius was only half that of S. or-yme, (5.3 unitslmg protein compared to 10.1 unitslmg protein, respectively), final activity following chromatography was 452.8 unitslmg protein, nearly identical to that of TABLE 1. Purification Sequence for Amylase From the Rice Weevil, Sitophilus oryzae Step Crude homogenate (Nh)2S04 Glycogen complex DEAE-SephaceF Volume (ml) Total proteir t (mg) Amylase” (units) Recoveryb (%) Specific activity‘ Fold purificationd 46.0 44.0 23.2 7.8 947.6 431.2 31.5 11.7 9,531 7,453 6,625 5,598 100 78 70 59 10.1 17.3 209.9 478.6 1.0 1.7 20.8 47.4 aOne unit of activity equals 1.0 mg maltose hydrate produced per minute at 30°C. bunits of activity at a given step divided by units in crude homogenate. “Units per milligram of protein. dSpecific activity at a given step divided by specific activity of crude homogenate. Baker and Woo 420 0 81 1.21 0 4 03 I 02 :z: 01 0 3 TUBE Fig. 1. Elution of glycogen-precipitated protein (A28o)and amylase (Aa) of S. oryzae from a DEAE-Sephacele ion exchange colum (1.6 x 13 crn) with a linear gradient of 0.4 M NaCl in 20 rnM tris-chloride, pH 7.5. Amylase was monitored by KI-iodine detection of residual starch. Tubes 94 to 102 were combined. TABLE 2. Purification Sequence for Amylase From the Granary Weevil, Sitophilus granarius Step Crude homogenate (NH4)2S04 Glycogen complex DEAE-SephaceF Volume (ml) Total protein (mg) Amylase" (units) Recoveryb 38.5 31.5 11.8 3.5 469.7 270.9 8.8 3.4 2,500 2,223 2,095 1,521 100 89 84 61 (YO) Specific activity' Fold purificationd 5.3 8.2 239.9 452.8 1.0 1.5 45.3 85.4 "One unit of activity equals 1.0 mg maltose hydrate produced per minute at 30°C. bunits of activity at a given step divided by units in crude homogenate. 'Units per milligram of protein. dSpecific activity at a given step divided by specific activity of crude homogenate. the purified amylase from S. oryzae. Final activity represented an %-fold purification with 61% of the original activity recovered. Absorbance coefficient (Al%,280 nm) of the purified amylase of S . granarius was 12.5. Electrophoresis Purified amylase from adults of S. oyzue consisted of two highly anionic isozymes that traveled with Rm values of 0.90 and 0.91 in a 7.5%polyacrylamide gel and 0.74 and 0.75 in a 10% gel, respectively. The amylases from S. o y m e were electrophoretically pure. Isoelectric points of the two amylase isozymes of S. oryzae were at pH 3.76 and 3.70. Purified amylase from adults of S. grunurius consisted of a single protein with R, 0.91 on the 7.5% gel and 0.75 on the 10% gel, identical to the fastest moving isozyme from S. otyzue. Except for a faint, diffuse, blue-staining band Purification and Amylases 421 at R , 0.85, the amylase sample from S. grunarius was electrophoretically clean. Isoelectric point of the amylase from S. grunurius was at pH 3.76. Both isozymes of S. oryme and the amylase of S. granarius had identical mobilities on the SDS-PAGE system (Fig. 2). Molecular weights of the enzymes were estimated to be about 56,000 for both species. The similar relative mobilities of the two isozymes of S. oryme on the anionic gels of both 7.5% and 10% concentration and the single band obtained with SDS-PAGE indicates that the amylase isozymes are charge isomers rather than size isomers. Kinetic Parameters Mean values (kSE) of Km with soluble starch as substrate were 0.173 k 0.005% for amylases from S. oryme and 0.078 f 0.003% for S. grunarius. Mean Vmax values for purified amylases at 30°C were 621 + 3 unitslmg protein for S. oryme and 592 f 11unitslmg protein for S. granarius. Expressed in terms of pmol maltoselminlmg protein, values were 1,730 pmollminlmg protein for amylases from S. oryzae and 1,640 pmollminlmg protein for amylase from s. grunurius. Amylases from both S. oryzue and S. granurius were activated by chloride (Fig. 3). Vmax for amylase increased 7.6-fold in S. oryzae and 6.5-fold for S. granarius in the presence of 20 mM NaC1. Presence of chloride did not affect the Km of S. oyzue but shifted the Km of S. grunarius slightly from about 0.09 to 0.07% starch. Fig. 2. Comparison of mobilities of SDS-treated amylases from S. oryzae and S. granarius with porcine pancreatic m-amylase and molecular weight standards. Lanes 1, 10, a-amylase, 15 pg; lanes 2, 3, Sigma standards, 5 and 10 &band; lanes 4, 5, amylase from S. oryzae, 5 and 10 pg; lanes 6, 7, amylase from S. granarius, 5 and 10 pg; lanes 8 , 9 , BioRad standards, 5 and 10 &band. Estimated M W of Sitophilus amylases was 56,000. 422 Baker and Woo Effect of Temperature Arrhenius plots of effect of temperature indicate that double energies of activation occur in purified amylases from both S. oyme and S. granarius (Fig. 4). Slopes of temperature effect between 10°C and 25°C for both species were identical with apparent Ea values 9.2 kcallmol. Between 25°C and 50°C activity slopes of the two species were not parallel and apparent Ea values were 6.3 and 7.5 kcal/mol for S. oyme and S. granarius, respectively. Comparative Amylase Levels in Sitophilus Specific activities of amylases were higher in larvae than in adults of S. oryzae and S. granarius. Activities in first-stage larvae (prior to feeding) were 10.7 f 1.5 and 7.3 & 1.1 unitslmg protein for S. oryzae and S. granarius, respectively. Activities in 7-8-day-old larvae were 24.3 & 0.8 and 14.6 k 2.2 + unitslmg protein, respectively. Highest amylase activity of 25.7 1.1units/ mg protein was found in fourth-instar larvae of S. oryzae. Amylase activities in larvae of S. oryzae at a given age were always significantly higher than those of larvae of S. grunarius. Only trace amounts of activity (< 0.1 unit/mg protein) were detected in pupae of mixed ages of either species. Activity in whole body extracts of newly eclosed adults (obtained from the artificial diet) was also low, 2.8 unitslmg protein for S. oyzue. Activities in whole body extracts of adults of S. oryzae, S. grunarius, and S. zeamais reared on whole wheat through 10 weeks of age are given in Figure 5. In S. oryzue, there was a slight trend toward higher specific activities as the weevils became older; however, amylase levels in all three species were variable from week to week. Significant differences in activity levels were 80 - 0 5. gronoriur 5. oryzoe 50 N o Chloride s * 40- Fig. 3. Effect of chloride (20 m M NaCI) on hydrolysis of soluble starch in 20 m M acetate, pH 5.0, by purified amylases from A) S. oryzae (7.6-fold increase in Vmax) and B) S. granarius (6.5fold increase in Vrnax). Purification and Amylases 423 3.0 Ea kcal/rnole 2.8 Temp 5. oryzae -- 5. gronariur 10'-25" 25"-50" 9.2 f 1 . 1 6 . 3 f 0 .4 9.2 f 0 - 9 7.5 f 0.5 2.6 > m 2 2.4 2.2 2 .o 3.1 3.2 3.3 '/K 3.4 3.5 3.6 (~103) Fig. 4. Arrhenius plots of effect of temperature on activity of purified amylases from S. oryzae and S. granarius. Double energies of activation were demonstrated for each species. 5. gronariut U 4 w 4 4- a 2- I n z 5. zeomair $ 04 0 1 2 3 4 5 6 7 8 9 1 0 ADULT AGE (Weeks) Fig. 5. Amylase activities of S. oryzae, S. granarius, and S. zeamais feeding on whole, soft red winter wheat at 28OC and 60% RH. Each point at each age interval is the mean of three replicates of ten weevils each for each species. 424 Baker and Woo found among species. Mean specific activities, excluding the initial (0 time) readings, for all values within the 10-week interval were 11.8 k 0.3, 4.3 k 2.0, and 1.5 k 0.2 unitslmg protein for S. o y m e , S. granarius, and S. zearnais, respectively. Based on protein, S. oyzue contained a mean of 2.9-fold and 7.8-fold more amylase activity than S. granarius and S. zearnais, respectively. S. grunarius contained 2.9 fold more amylase than S. zearnais. Mean activities of whole body extracts expressed on a fresh weight basis were 725 k 15, 237 & 9, and 90.2 k 8.5 milliunitslmg fresh weight for S. oryzae, S. granarius, and S. zeurnais, respectively. Thus, based on fresh weight, S. o y m e contained threefold and eightfold more amylase activity than S. granurius and S . zearnais, respectively. S. granarius contained 2.6-fold more amylase than S. zearnais. Adults of S. zearnais were largest (mean fresh weight of about 3.8 mg compared to 2.8 mg for S. granarius and 2.2 mg for S. oyzue throughout the 10week period) but contained extremely low amylase levels relative to the two other species. DISCUSSION Properties of Amylases Procedures used to purify amylases from insects include glycogen precipitation followed by ion exchange chromatography [ZO],coprecipitation with amylopectin , and affinity gel chromatography . The glycogen precipitation and anionic ion exchange chromatography purification sequence used for S. oryzae and S. granarius resulted in extremely active preparations with good yields. Comparisons of activities of purified amylases from insects are complicated since definitions of "units" of activity vary in each case. Activity of amylase from larvae of Callosobruchus chinensis (L.) after a glycogen precipitation step was 590 unitslmg protein, 1 unit equivalent to 1 mg maltosell0 min at 37°C . Disregarding temperature, this activity corresponds to 59 "Sitophilus" units, which is 3.5- and 4-fold lower than activity of amylases from S. oryzae and S . grunarius, respectively, at the equivalent step in the purification procedure. Activity of electrophoretically pure amylase from Tenebrio molitor L. was 859 unitslmg protein, 1 unit equivalent to 1 pmol maltoselmin at 37°C . If activity units for amylases from S. oyzue and S . granarius are expressed in pmol maltoselmin, final activities at 30°C were 1,300 and 1,250 unitslmg protein, respectively. Thus, the purified amylases from Sitophilus are apparently much more active than that from C. chinensis and are also more active than the purified amylase from T. rnolitor. Molecular weights of mammalian and bacterial amylases range from about 40,000 to 59,000 . Molecular weights of insect amylases are comparable. Amylase from T. rnolitor was a single polypeptide chain with an SDS-PAGE estimated molecular weight of 68,000 . Estimates of the molecular weight of purified amylase from Bornbyx mori were 57,000 by SDS-PAGE and 47,000 by guanidine column chromatography on Sepharose . Molecular weight estimates of 56,000 for amylases of both S. oryzae and S . grunarius by SDSPAGE are comparable to these purified enzymes. Amino acid analyses of the Sifophilus amylases have not been determined. Absorbance coefficients of aamylases at 280 nm are relatively high because of high concentrations of Purification and Amylases 425 tryptophan and tyrosine . However, A*%values of 13.3 and 12.5 for S. oy m e and S. granarius, respectively, were about half those calculated for porcine pancreatic amylase and human salivary amylase. Isoelectric points of pH 3.70 and 3.76 for the isozymes of S. oryme and pH 3.76 for the single amylase of S. grunurius were slightly lower than that of T. molifor (pH 4.0)  and generally lower than isoelectric points (range pH 4.2 to 5.7) for most other amylases . The highly anionic nature of the Sitophilus amylases at neutral to slightly alkaline pH apparently accounts for their fast mobility on the polyacrylamide gels. Amylases from larvae of S. grunuritds and S. zearnuis were activated by 0.1 mM NaCl in buffered starch solution [l]. Presence of C1- also resulted in substantial increases in Vmax for the purified enzymes from adult Sitophilus. Chloride generally activates insect amylases [22,24] but C1- inhibited amylase from C. chinensis . Ca2+ stabilized the larval amylases of S . grunarius and S. zeamais against thermal inactivation [l]. The role of Ca2+ in the purified amylases from adults has not been determined but presumably this divalent cation has a similar function. Purified amylases from both S. oryzue and S. grunarius have double energies of activation, 9.2 Kcallmol between 15°C and 25°C and 6.3 and 7.5 Kcallmol between 25°C and 50"C, respectively. Most mammalian and bacterial amylases have single Ea values . However, double energies of activation were shown for the amylase from the bacterium Pseudornonas saccharophilu, 14.4 Kcallmol between 0°C and 15°C and 8.5 Kcallmol between 15°C and 40°C . Although there have been only a few such studies with insects, insect amylases with both single and double energies of activation have been demonstrated. Single Ea values were shown for C. chinensis, 7.3 Kcallmol between 10°C and 60°C  and Rhynchosciara umericanu, 4.8 Kcal/mol between 12°C and 35°C . Double Ea values were calculated for the purified amylase from B. mod, 17 Kcallmol between 0°C and 25"C, and 3.8 Kcallmol between 25°C and 40°C . Amylases from larvae of S. grunurius and S. zeurnuis were endoamylases of the a-amylase type [l]. Km values for soluble starch of larval amylases were 0.077% for S. grunurius and 0.13% for S. zeumais. The Km value for starch for purified amylase from adult S. granarius was 0.078%, nearly identical to that of the larval amylase. Single isozymes were found in both larvae and adults of S. granurius. Km for starch for adults S. oryme was 0.173%, somewhat higher than the Km determined for larvae of S . zeurnuis. Both S . o y m e and S. zeamais have two amylase isozymes that are resolved on polyacrylamide gels. Amylase from Sitophilus have a higher affinity for starch than the amylase from C. chinensis (Km of 0.23%)  but the amylase of S . oryme has a Km similar to that of T. rnolifor (Km of 0.18%) . Adaptive Significance of Amylase Levels in Sitophilus Although amylase of S. oryzae and S . grunurius were purified to the same specific activity, total amylase levels in S. oqzue were significantly higher than those of S. grunurius in developing and adults stages. These high amylase levels may provide a selective advantage to S. oryme even though 426 Baker and WOO the Km for starch for S. oyme (0.173O/0) was much higher than that of S. granarius (0.078%). When reared on wheat, the capacity for increase of S. o y m e is greater than that of both S. zeumuis  and S. grunurius [27,28]. In addition, S. oryzue is more efficient than S. grunurius in utilizing whole wheat [4,29] or a starchbased meridic diet  as food sources and out-competes S. granarius in competition experiments . A combination of behavioral and physiological responses, including the differential presence of symbiotic microorganisms in these species , probably accounts for faster population development by S. oryzae. Nevertheless, the presence of significantly higher levels of aamylase in s. otyzae may also confer an adaptive advantage for this species by negating the effect of a-amylase inhibitors when feeding on wheat. Amylase levels demonstrated in adults of S. oyzue, S. granarius, and S. zeurnais do not always correlate to development profiles of these species on different cereals or the relative susceptibility of different cereals to weevil attack. Although S. mmuis has relatively low levels of amylase, this species develops faster than S. grunurius on triticale, wheat, barley, and maize . However, barley and maize do not contain the a-amylase inhibitors  so insect amylase levels may not confer any advantage or disadvantage when Sitophilus feed on these cereals. On the other hand, triticale does contain an amylase inhibitor  but softness of the endosperm rather than biochemical composition of this cross between wheat and rye may be more important in its susceptibility to weevil attack . A number of the low molecular weight, water soluble albumins found in wheat inhibit amylases . The albumins form reversible complexes with the amylase from T. rnolifor  and are resistant to proteolytic degradation by midgut proteinases from this species . Since midgut proteinase levels in Sitophilus are very low , detoxification of ingested amylase inhibitor by proteolytic degradation also seems unlikely in these species. Thus, the relatively high amylase levels in Sitophilus and the significantly higher levels in S. ovyzae may be an adaptation not only to high dietary levels of starch but also to counter the effect of ingested inhibitors from cereals by actually flooding the inhibitors with enzyme to thereby allow normal digestive mechanisms to proceed. LITERATURE CITED 1. Baker JE: Properties of amylases from midguts of larvae of Sitophilus zeamais and Sitophilus grunurius. Insect Biochem 13, 421 (1983). 2. Baker JE, Woo SM: Unpublished observations (1984). 3. Baker JE, Woo SM, Byrd RV: Ultrastructural features of the gut of Sitophilus gvanarius (L.) (Coleoptera: Curculionidae) with notes on distribution of proteinases and amylases in crop and midgut. Can J Zool 62, 1251 (1984). 4. Campbell A, Singh NB, Sinha RN: Bioenergetics of the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae). Can J Zool 54, 786 (1976). 5. D’Appolonia BL, Gilles KA, Osman EM, Pomeranz Y: Carbohydrates. In: Wheat Chemistry and Technology. Pomeranz Y, ed. American Association of Cereal Chemists, St. Paul, Minn., pp 301-392 (1971) 6. Silano V, Furia M, Gianfreda L, Nacri A, Palescendolo R, Rab A, Scardi V, Stella E, Valfre F: Inhibition of amylases from different origins by albumins from the wheat kernel. Biochim Biophys Acta 391, 170 (1975). Purification and Amylases 427 7. Kneen E, Sandstedt RM: An amylase inhibitor from certain cereals. J Am Chem SOC65, 1247 (1943). 8. Applebaum SW: The action pattern and physiological role of Tenebrio larval amylase. J Insect Physiol 20, 897 (1964). 9. Applebaum SW, Konijn AM: The utilization of starch by larvae of the flour beetle, Tribolium custuneum. J Nutr 85, 275 (1965). 10. Yetter MA, Saunders RM, Boles HP: a-Amylase inhibitors from wheat kernels as factors in resistance to postharvest insects. Cereal Chem 56, 243 (1979). 11. Bernfeld P: Amylases, a and 0.Methods Enzymol 1, 149 (1955). 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 293, 265 (1951). 13. Robyt JF, Whelan WJ: The a-amylases. In: Starch and Its Derivatives. Radley JA, ed. Chapman and Hall, London, pp 430-476 (1968). 14. Loyter A, Schramm M: The glycogen-amylase complex as a means of obtaining highly purified a-amylases. Biochim Biophys Acta 65, 200 (1962). 15. Davis BJ: Disc electrophoresis-11. Method and application to human serum proteins. Ann NY Acad Sci 121, 404 (1964). 16. Baker JE: Digestive proteinases of Sitophilus weevils (Coleoptera: Curculionidae) and their response to inhibitors from wheat and corn flour. Can J Zol60, 3206 (1982). 17. Baker JE: Application of capillary thin layer isoelectric focusing in polyacrylamide gel to the study of alkaline proteinases in stored-product insects. Comp Biochem Physiol 71B, 501 (1982). 18. Baker JE, Mabie JA: Growth and development of larvae of the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), on natural and meridic diets. Can Entomol 105, 249 (1973). 19. Baker JE, Mabie JA: Growth responses of larvae of the rice weevil, maize weevil, and granary weevil on a meridic diet. J Econ Entomol66, 681 (1973). 20. Podoler H, Applebaum SW: The a-amylase of the beetle Cullosobruchus chinensis-Purification and action pattern. Biochem J 122, 317 (1971). 21. Kanekatsu R: Studies on further properties for an alkaline amylase in the digestive juice of the silkworm, Bombyx mori. J Fac Textile Sci Techno1 76 (Series E, No. 9), (1978). 22. Buonocore V, Poerio E, Silano V, Tomasi M: Physical and catalytic properties of a-amylase from Tenebrio rnolitor L. larvae. Biochem J 253, 621 (1976). 23. Buonocore V, Poerio E, Gramenzi F, Silano V: Affinity column purification of amylases on protein inhibitors from wheat kernel. J Chrom 114, 109 (1975). 24. Terra WR, Ferreira C, DeBianchi AG: Action pattern, kinetical properties and electrophoretical studies of an alpha-amylase present in midgut homogenates from Rhynchosciuru americanu (Diptera) larvae. Comp Biochem Physiol56B, 201 (1977). 25. Markovitz A, KIein HP, Fischer E: Purification, crystallization, and properties of the a amylase of Pseudomonus sacchurophila.Biochim Biophys Acta 29, 267 (1956). 26. Birch LC: Experimental background to the study of the distribution and abundance of insects. I. The influence of temperature, moisture and food on the innate capacity for increase of three grain beetles. Ecology 34, 698 (1953). 27. Evans DE: The capacity for increase at low temperature of several Australian populations of Sitophilus o y m e (L.). Aust J Ecol2, 55 (1977). 28. Evans DE: The capacity for increase at a low temperature of some Australian populations of the granary weevil, Sitophilus grunarius (L.). Aust J Ecol2, 69 (1977). 29. Singh NB, Campbell A, Sinha RN: An energy budget of Sitophilus oryzae (Coleoptera: Curculionidae). Ann Entomol SOCAm 69, 513 (1976). 30. Baker JE: Differential net food utilization by larvae of Sitophilus oryme and Sitophilus grunurius. J Insect Physiol20, 1937 (1974). 31. Tripathi RL, Hodson AC: Factors responsible for the competitive superiority of the rice weevil, Sitophilus oryzue Linn. over the granary weevil, Sitophilus grunurius Linn. Indian J Entomol43, 1(1981). 32. Buchner P: Endosymbiosis of Animals With Plant Microorganisms. Interscience, New York, pp 1-909 (1965). 33. Dobie P, Kilminster AM: The susceptibility of triticale to post-harvest infestation by Sitophilus zeurnuis Motschulsky, Sitophilus o y m e (L.) and Sitophilus grananus (L.). J Stored Prod Res 14, 87 (1978). 428 Baker and Woo 34. Kneen E, Sandstedt RM: Distribution and general properties of an amylase inhibitor in cereals. Arch Biochem Biophys 9, 235 (1946). 35. Saunders RM: a-Amylase inhibitors in wheat and other cereals. Cereal Foods World 20, 282 (1975). 36. Bedetti C, Bozzini A, Silano V, Vittozzi L: Amylase protein inhibitors and the role of Aegilops species in polyploid wheat speciation. Biochim Biophys Acta 362, 299 (1974). 37. Buonocore V, Gramenzi F, Pace W, Petrucci T, Poerio E, Silano V: Interaction of wheat monomeric and dimeric protein inhibitors with a-amylase from yellow mealworm (Tenebrio rnolitor L, larva). Biochem J 187, 637 (1980).