Oct 2, 1980 - ated, and subjected to guanidinium chloride gel filtration. ..... Wcthodrl; (01 electrophoretogram Of natlve crystal dissolved In 12 (wtfvoll radium ...
THEJOURNALOF BIOLOGICALCHEMISTRY Val. 256, No. 6. Issue ofMarch 25, pp. 3000-3004,1981 Printed in U.S.A.
Purification and Characterizationof the Entomocidal Protoxinof Bacillus thuringiensis* (Receivedfor publication, October2, 1980, andin revised form, November20, 1980)
Lee A. Bulla, Jr.#&Karl J. Gamer$?, DavidJ. Coxl, Berne L. Jones+,Loren I. Davidsons, and George L. Lookharts From the $United States GrainMarketing Research Laboratory, Agricultural Research, Science and Education Administration, United States Department of Agriculture and the §Division of Biology, Kansas State University, Manhattan, Kansas 66502 and the 1[ Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
MATERIALS AND METHODS’ A procedure forpurifying the insecticidal parasporal protoxin of Bacillus thuringiensis and a description of RESULTS its biochemical and biophysical properties is provided. generate a funcMild alkali titration was necessary to Isoelectric Point and Effect of p H and Time on Crystal tional protoxin in a soluble form, and anion-exchange Solubilization-Crystals, isolated by buoyant density centrifchromatography was used to remove contaminating ugation in Renografii gradients (22) and washed thoroughly cytoplasmic proteases that are nonspecifically bound in distilled HzO, were titrated with 1 N NaOH until solubilito whole native parasporal crystals. Polyacrylamide zation was observable by microscopic examination and by an gel electrophoresis, gel filtration chromatography,and increase in absorbance at 280 nm of the aqueous suspension. meniscus depletion sedimentation equilibrium analysis For a 0.4% suspension (w/v), complete dissolution occurred revealed an apparent molecular weight for the protoxin of 1.34 X 10’. The only NHz-terminal residue found was when the sodium hydroxide concentration reached 13.5 ~ l methionine. The soluble protoxin w a s 2.5 times more and the pH wasabout 12. Approximately 4 pmol of alkali were toxic to insect larvae than w a s the parasporal crystal. required to dissolve 1 mg of crystal, equivalent to 400 mol of At alkaline pH the protoxin slowly converted toa low hydroxide ion/mol of subunit (apparent M , = 1.34 X lo5). molecular weight toxin (apparent M, = 6.8 X lo4). The This value was in good agreement with the potential number molar specific toxicities of the protoxin and toxin were of ionizable side chains in the crystal (1). At pH 10,11, and 12, the solubility limits were 0.2, 1.4, and 4 mg/ml, respectively. identical. Apparently, only the highly charged anionic subunit of the crystal was completely soluble. These results are consistent with a crystal subunit that has an isoelectric point near Bacillus thuringiensis is a gram-positive, aerobic, spore- physiological pH. A PI of 7.2 0.1 was determined by isoelecforming bacterium that synthesizes an intracellular parasporal tric focusing on polyacrylamide. glycoprotein crystal during the sporulation cycle (1, 2). The Treatment of crystals with excess alkali reduced the yield crystal, which represents 20-30% of the cell dry weight, is of soluble protoxin. For example, no biologically active protein toxic to lepidopteran insects (3). In addition to itsinsecticidal was recovered from crystals treated with 1 N NaOH for 20 h. properties, this inclusion body has been reported to cause Time of incubation also had an effect. Dissolution was comtumor regression (4, 5) and to enhance the overall immune plete in 13.5 m~ NaOH after 3 h; however, about 40% of the response in rats (6). subunit remained in an aggregated state as revealed by chroThe glycoprotein is a protoxin that is activated after inges- matography on DEAE-Bio-Gel A (Fig. lA, arrow denotes tion by an insect susceptible to thetoxic product (3). Although dissociated monomer). Only after 4-5 h was the monomer information is available aboutthe physical and chemical almost completely dissociated (fraction 48, Fig. 1B). When properties of the crystal (1, 7-17), little is known about its in incubated for periods greater than 24 h at pH 12, the subunit vivo function in the bacterium or about the mechanism of yield decreased such that after 168 h, only 30%of the subunit toxicological action (18-21). The primary reason for this lack remained (Fig. IC). Biological activity of the alkali-treated of understanding is that a reproducible method has not been crystals correlated well with the yield results obtained by ionavailable for purifying the crystal and rendering the protoxic exchange chromatography. subunit soluble in a relatively stable forrh. Here we report a Physical Characterization-The homogeneity of the proprocedure forpurifying the protoxin as well as toxin and toxin, solubilizedfor 4 h in alkali, was further analyzed by gel describe some of their chemical and biological properties. filtration on Sepharose CL-4B at pH 8.4. A nearly symmetrical peak was obtained with apparent molecular weight of 1.34 * This workwassupportedbyGrantPCM7907591from the National Science Foundation. ContributionNo. 80-125-5,Department 0.20 X lo5 (chromatogram not shown). When this preparation
*
*
of Biochemistry, Kansas Agricultural Experiment Station, Manhattan, KS 66506. Cooperative investigation between Agricultural Research, Science andEducationAdrmnistration, United States Departmentof Agriculture, and the KansasAgriculturalExperiment Station, Manhattan, KS. Mention of a trademark or proprietary product does not imply its approval to the exclusion of other products that may also be suitable. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’ Portions of this paper (including “Materialsand Methods,”Figs. 1-5, Tables I and 11, andadditionalreferences)arepresented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopiesare available from the Journal of Biological Chemistry, 9650 RockviUe Pike, Bethesda, Md. 20014. Request Document No. 80 “2085, cite authors, and include a check for $5.20per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
3000
l
~
Entomocidal Protein was examined by ultracentrifugation under nondenaturing conditions, a time-dependent fringe displacement occurred that indicated that solute was changing irreversibly during the run. Therefore, a sample of the protoxin was reduced and S-carboxymethylated in 6 M guanidinium chloride. A t sedimentation equilibrium under denaturing conditions, the modified protoxin gave a molecular weight of 1.34 x lo5 (Fig. 2). The data plot was essentially linear over the entire solution column. The suggestion of downward curvature at thehighest concentrations was to be expected for ahomogeneous solute in concentrated guanidinium chloride because, inthis solvent, protein solutions show considerable thermodynamic nonideality (23). The molecular weight of the alkylated protoxin also was determined by guanidinium chloride gel fdtration (24).Eightyfive % of the protein applied to thecolumn eluted at an elution volume whichcorresponded to thatof a protein whose molecular weight is 1.3 f 0.2 x lo5. Appropriate fractions of the material were combined, dialyzed, lyophilized, and electrophoresed on a sodium dodecyl sulfate polyacrylamide gel. A single band was observed at a molecular weight of1.34 -+ 0.20 X IO5 (Fig. 3A, band I ) . This electrophoretic result was in contrastto thatobtained when the parasporal crystal was not alkali-solubilized, alkylated, and subjected to guanidinium chloride gel filtration. When the crystal was dissolved in denaturing and reducing agents as described previously (1)and directly electrophoresed on polyacrylamide (Fig. 3B), a major band (band I ) at an apparent molecular weight of1.34 X lo5 was observed, together with several minor bands (bands 2, 3, and 4). AII but one of the proteins (band 4 ) were larger in molecular sizethan the major protein (band I ) . The heavier components (bands 2 and 3) apparently arose from molecular association of the glycoprotein because their electrophoretic mobilities corresponded to those expected of oligomeric forms of the 1.35 X lo5”, subunit. Proteolytic and Insecticidal Activities-Previously, we reported that casein hydrolytic activity was associated with alkali-solubilized crystal (I). It seemed reasonable that because extracts of sporulating bacilli have particularly high proteolytic activity (25), the proteolysis associated with the protoxin might be due tocontamination from either extracellular or intracellular proteases. This probably was the case since all of the casein hydrolytic activity was removed from the alkali-solubilized monomer by chromatography on DEAEBio-Gel A (Fig. 4). The crude extracts had 15-30 times greater protease-specific activity than the DEAE-Bio-Gel A-purified subunit. The purified subunit still was fullyactive biologically. The estimated 50 per cent lethalconcentration values (based on measurable protein) for the DEAE-Bio-Gel A-purified material, tested against the tobacco hornworm, was approximately 2.5 times more toxic than the native crystal (Table I). This result indicated that casein proteolytic activity was not essential for toxicity as had been suggested previously (1). After casein hydrolytic activity had been removed, the subunit was still somewhat unstable. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the purified subunit that had been held at 28°C and pH 8.4 for 72 h (Fig. 5A) showed not only the M , = 1.34 x lo5 subunit (band I ) , but it also revealed a lower M , component (apparent M , = 6-7 x lo4; band 2) which co-migrated with the tracking dye. The latter protein, referred to as toxin, was isolated by anionexchange chromatography and possessed virtually the same mol/cm2) as the protoxin toxicity on a molar basis (1.6 X mol/cm2) toward the tobacco hornworm (Table (2.0 X I). Fig. 5B shows a sodium dodecyl sulfate polyacrylamide gradient gel of the toxin purified by DEAE-chromatography.
of
B. thuringiensis
3001
A single band was observed whose mobility corresponded to that of a protein with an apparent molecular weight of 6.8 X
lo4.
Chemical Composition-The amino acid compositions of the parasporal crystal and protoxin are presented in Table 11. For comparison the compositions were normalizedto amolecular weight of 6.8 X lo4. The parasporal crystal and protoxin had similar amino acid compositions. The minor differences probably were due to contaminating proteases associated with the crystal. Both compositions were characterized by large amounts of aspartic acidor asparagine, glutamic acidor glutamine, and arginine. Analysis of the NHz-terminal residue by Edman degradation using thin layer and high performance liquid chromatography revealed methionine as the only detectable amino acid in both the solubilized subunit and native crystal. No hexose analysis was conducted on the solubilized subunit, but we assume on the basis of identical physical behavior, that the carbohydrate composition is the same for both (3.8% glucose and 1.8% mannose;Ref. 1). Solubilized protoxin produced a positive anthrone reaction. Furthermore when 1 mCi of ~-[2-~H]mannose was added to a sporulating culture of B. thuringiensis and the crystals were subsequently isolated and solubilized by alkali titration (see “Materials and Methods”), the solubilized subunit was radioactive, indicating glycosylation had occurred, probably via mannose-l-P04.No sialic acid derivatives or amino sugars were detected using thiobarbituric acid and ion-exchange chromatography, respectively. The toxin had a very different amino acid composition when compared tothe protoxin and, therefore, is truly unique (Table 11). Major differences were found in the amounts of tryptophan, aspartic acid, serine, alanine, valine, and methionine. Periodic acid-Schiff staining of the toxin purified by polyacrylamide gel electrophoresis revealed that it also is glycosylated. DISCUSSION
We have found that thesolubility and toxicity of the parasporal crystal of B. thuringiensis varies with both pH and time. Nishiitsutsuji-Uwo et al. (26) obtained similar results with regard to pH using another strain (subspecies aizawai) of the bacterium. Maximum solubility of the kurstakicrystal occurred about 5 h after the crystal was titrated with 400 eq of base. The subunit was stable for several hours thereafter, but then began to degrade into smaller fragments with a concomitant loss in insecticidal activity. Reaggregation also occurred, especially after the pH was lowered to near neutrality. These findings are all consistent with the experimentally determined PI of the protoxin, pH 7.2. Previously, we reported a range of molecular weights for the native subunit from 0.9-1.3 X lo5, depending on the method usedforsolubilization and for size determination, When we examined material solubilized by the mild titration procedure detailed in this report, all molecular weight determinations gave essentially the same result of 1.34 X lo5. Also, a single NH2-terminal residue was detected in quantitative yield. The crystal protein may be an intact product of translation since methionine was the NHn-terminal residue. Preparations that we used previously apparently were contaminated with small amounts of lower molecular weight components. This probably was the result, in part, of a contaminant protease that was not fractionated away from the crystal by the density centrifugation method. Now, by ion-exchange chromatography of the akali-solubilized crystal, this protease has been excluded.The protease was probably an intracellular protease, generated during sporulation, that bound nonspecifically to thecrystal. Previously, Chestukina et al. (8)detected
Entomocidal Protein
3002
of B. thuringiensis Acknowledgments-We are grateful to J. Hubbard, J. Schesser, and D. B. Cooper for expert technical assistance, and to J. Nordin, Department of Biochemistry, University of Massachusetts, Amherst, and to J. Iandolo, Division of Biology, Kansas State University, for helpful discussions. REFERENCES
SR SR MWOpp=I. 34 x 1 0'
FIG. 6 . Schematic diagram of the behavior of parasporal crystal subunit in solution.
several proteases associated with parasporal crystals derived from B. thuringiensis subsp. galleriae and infectum. We also have isolated a toxic protein (apparent M, = 6.8 X IO4) that is generated from protoxin upon prolonged incubation (4-6 days) at slightly alkaline pH. This glycoprotein remained toxic at room temperature for several months at neutral pH. It was the smallest toxic component that we found and any furtherbreakdown was detrimental to toxic activity. When compared to the protoxin, the 68,000-Mrpolypeptide had very similar toxicity (50 per cent lethal concentration E 2X mol/cm2) and, like the protoxin, was2.5 times more insecticidal than thenative crystal. Apparently, alkali titration of crystal and subsequent incubation of protoxin is an efficient in vitro method for toxin production. Fig. 6 is a schematicdiagram that summarizes the behavior of the protoxin and toxin in solution.As depicted, the scheme points out certain precautions that are necessary to preserve both subunit structure and biological activity. The crystal (An) is made up of many subunits that are dissociated in native conformation (nA)by mild alkali titration (reaction 1). Reaggregation OCCLUS slowly (reaction 2), especially at pH 5 12; also, the subunit may break down to smaller fragments (reaction 4). Degradation may be further stimulated by contaminatingproteases that co-fractionate with the crystals during isolation. To properly characterize the physical and chemical properties of the subunit, it was necessary to use the material within several hours of preparation. Otherwise, the subunit must be stablized by placing it in a denaturingsolvent and alkylating the disulfide linkages (reactions 6 and 7). The toxin can be generated by prolonged incubation at slightly alkaline pH (reaction 3). Whether this reaction is similar to the in vivo mechanism of activation (reaction 5) is not known. We now are investigating further the chemical and physical properties of the toxin and its mechanism(s) of activation.
1. Bulla, L.A., Jr., Kramer, K. J., and Davidson, L. I. (1977) J . Bacteriol. 130, 375-383 2. Bechtel, D.B., and Bulla, L. A., Jr. (1976) J. Bacteriol. 127, 1472-1481 3. Bulla, L. A., Jr., Bechtel, D. B., Kramer, K. J., Shethna, Y. I., Aronson, A. I., and Fitz-James, P. C. (1980) CRC Crit. Reu. Microbiol. 8, 147-204 4. Prasad, S. S. S. V., and Shethna, Y. I. (1974) Biochim. Biophys. Acta 362,558-566 5. Prasad, S. S. S.V., and Shethna, Y. I. (1976) Antimicrob. Agents Chemother. 10,293-298 6 . Prasad, S. S. S. V., and Shethna, Y. I. (1975) Biochim. Biophys. Res. Commun. 62,517-523 7. Seki, T., Nagamatsu, M., Nagamatsu, Y., Tsutsui, R., Ichimaru, T., Watanabe, T., Koga, K., and Hayashi, K. (1978) Sei. Bull. Fac. Agric. Kyushu Uniu. 1, 19-24 8. Chestukhina, G. G . , Kostina, L. I., Zalunin, I. A., Kotova, T. S., Katrukha, S. P., Kuznetsov, Y. S., and Stepanov, V. M. (1977) Biokhzmiya 42,1660-1667 9. Prasad, S. S. S. V., and Shethna, Y. I. (1974) Biochim. Biophys. Acta 362,558-566 10. Glatron, M.-F., Lecadet, M.-M., and Dedonder, R. (1972) Eur. J. Biochem. 30, 330-338 11. Herbert, B. N., Gould, H. J., and Chain, E. B. (1971) Eur. J. Biochem. 24,366-375 12. Akune, S., Wanatabe, T., Mukai, J., Tsutsiu, R., and Abe, K. (1971) Jpn. J. Med. Sei. Biol. 24, 57-59 13. Sayles, V. B., Jr., Aronson, J. N., and Rosenthal, A. (1970) Biochem. Biophys. Res. Commun. 41, 1126-1133 14. Holmes, K. C., and Monro, R. E. (1965) J. Mol. Biol. 14,572-581 15. Pendleton, I. R. (1968) J. Appl. Bacteriol. 31,208-214 16. Fitz-James, P., and Young,I. E. (1958) J. Biophys. Biochem. CytoZ. 4,639-660 17. Hanney, C. L., and Fitz-James, P. (1955) Can. J. Microbiol. 1, 694-710 18. Heimpel, A. M., and Angus, T. A. (1959) J. Insect Pathol. 1,152170 19. Murphy, D. W., Sohi, S. S., and Fast, P. G. (1976) Science 194, 954-956 20. Travers, R. S., Faust, R. M., and Reichelderfer, C. F. (1976) J . Znuert. Pathol. 28,351-356 21. Griego, V. M., Moffett, D., and Spence, K. D. (179) J. Insect Physiol. 25,283-288 22. Sharpe, E. S., Nickerson, K. W., Aronson, J. N., and Bulla, L. A. (1975) Appl. Microbiol. 30,1052-1053 23. Munk, P., and Cox, D. J . (1972) Biochemistry 11,687-697 24. Mann, K. G., and Fish, W. W. (1972) Methods Enzymol. 26, Part C, 28-42 25. Doi, R. H. (1972) Curr. Top. Cell. Regul. 6, 1-20 26. Nishiitsutsuji-Uwo, J., Ohsawa, A., and Nishimura, M. S. (1977) J. Znuert. Pathol. 29, 162-169 Additional references are found on p. 3003.
Entomocidal Protein
of
B. thuringiensis
3003
Sepharose CL-48 e1 I100to 200 npsh; Phamacla) was e q u i l i b r a t e d w l t h 6 H guanidinrum c h l o r i d e i n 0.05 H 2-~N-morphollno)ethanerulfo"i~ acid (PES) (pH 6.0). The g e ls l u r r y was poured t o a column bed height of 85 cm i n a glass column (Pharmacia, 1.5-cm inner d i a n e t e r ) . The agarose bed we1 f u r t h e r e q u i l i b r a t e d by passing two column ~ o l u m sof the abew buffer f l w r a t e of 4mlfh. The w i d and i n c l u s i o n volumer were determined throughthe column a t a e usinq bluedextran 2000 and L3Hl-labeleddiisopropylphorphorofluaridate.rerpectiuely. The E. t h u r i n iensis c w t a l p r o t e i n was c a n p a d u n d e r elution behavior of 8-carbox-thylated identical c o n d i t i o nw i t ht h a to fp r o t e i n m i h t I t a n d w d s ( 2 rnq/0.4 m 1 i n c l u d i n g myosin IEW-2.2 x 10'). beta-galactosidase 11.3 x 105).phospholylase A (9.4 x loll. and g a m globulin subunit (heavychain, 5 I: 10 1.
r o t a r y a g i t a t i o n a t 250 rpn
The e l e c t r o p h o r e t i c m b i l l t i e r Of t h e f o l l a l n g r m l e c u l a r w e i g h t standards were compared with that of protoxin i n 51 plyacrylamidegelscontaining0.1% SO8 buffered i n phosphate (pH 7.0): myosin (Hy=Z.2x 108 kta-gdlaCtOSidase(1.3 I 105). PhOIPhorylls A (9.4 X l o 4 ) , bovine serum albumin 16.8 x 10dj. a m g l o b u l i nr u b u n i t (heavy chain. 5 x l o 8 ) , ovalbumin 14.5 x 104). and pepsin (3.5 x 1047. Anion exchdn e Chl'mato ra h : SalubillzedCrystal was dialyzed a a i n r t 20 mM NaH2P04 b u f f e r pH 7.5 a i d Subreque%l! i p p l i e d t o a colunn of DEAE-810-Gel 4 q1.5 x 40 en) equilib!ated v i t h 20 rM NA PH O (pH 7.5) and fractions I 3 n l ) O f e f f l u e n t *ere collected. A f t e r washingthe column m f h c o l ~ m nvolunes of buffer. I gradient O f 0 t o 0 . 4 M NaCl i n the phosphate buffer Wdl applied to elute the protoxin Subunit.
t
REFERENCES [References 11-26) are I n theparent 27. P o l acr l a m i d ee le l e ~ t r o p h o r e ~ 1 1 :E l e c t r o p h o r e s i s in p o l y a c w l m i d e containing 0.1% 508 was k r f h bygthe methods o f U e k r e t a l . (28) and Omstein and D a v i s (29). Gels were Stained with Caomarsie b r i l l i a n t blue (0.251, r t f v a l ) and destainedbywashing in methanol-aceticacid-water(25:7.5:62.5.volfvolfval)for 16 t o 20 h. Glycoprotein(vicinal hydroxyl g r w I ) was v i s u a l i z e d d i r e c t l y on gels by rtainlng with periodate-Schiff reagent (PAS. ref. 30!, Beforestaining,thegels were incubatedovernight i n a mmrwre O f 251; isopropylalcohol and IO% a c e t i c a c i d t o f i x t h e p r o t e i n s and r m v e 80s. They were f u r t h e r preconditionedNith0.51 sodium arsenite and 51aceticacid.Destaining was accomplished by soaking the yclr for 16 h i n a s o l u t i o n of 0.1% sodium m t a b i l u l f i t e and 0.01 N HCI. O e n r i t o w t e r traCingS Of the gels were obtained with a gel scanning attachment Of a G i l f o r d 250 r p e c t r o p h e t m t e rI G i l f o r dI n s t r u m e n t s Lab. I n c . , Oberlln,Ohio). Gels stainedwith Coomassie b r i l l i a n t b l u e were scanned a t 550 nm and oels rtdined wlth Oeriodate-Schiff I s o e l e c t r i c focusing (31) yds w C O ~ l i S h e d b y h i g h Pesolution gradient g e l s l a b electrophoresis ( P h a m c i a Gel Electrophoresis Apparatus GE-41 using pH 5 t o 9 Amphaline carrier smpholyter (2%. ufv. LKB). Electrofocuringoccurred i n 10-M g e l sa t 15'Cand 1000 V f o r 2 1/2h.Approximately ZOO yg of s o l u b i l i z e d p r o t e i n y i e l d e d bands t h a t gaYe optimal Itdlning. Protein detemination: Protein concentration was estimatedbythe L a r y procedure (32) and by absorbance a t 2wI m using an e x t i n c t i o n c o e f f i c i e n t of 1.0 absorbance U n i t equal t o 11 mg Of proteinper m l i n a 1 cm c e l l Amino a c i d analysis: Samples were analyzed On 1 8eCbdn 12oC analyzerafterhydrOlylis f o r z T K i n 6 N Hc1 c m t a i n i w 0.1% phenol. The amunts o f l a b i l e amlno a d d s were calculated by e x t r a m l a t i o n o f a n a l y s e s for. 24,48. and 72 h w r s t o zero tim. C y r t l n e and cysteine w r e detemined as cysteic acid and m t h i a n i n e a s the sulfone derivative a f t e r p e r f a m l c acid oxidation ( 3 3 ) . Tryptaphan was quantitated in sampler hydrolyzed in 3 H ptoluenesulfonicacidcontalnin a m a l l a m n t oftryptamlne. The h y d r o l y r l r was c a r r i e d Out f o r llo'c q341. 24 aht
1"
Protein hydrolysater e r e a l s o analyzed as their 0-phthaldlaldehyde derivatives (35) using a h i g h perforRdnCe l i q u i d Chronatography i y l t e m c b n s i s t i n g O f a Uarian 5020 W V , a Rheodyne7120 injectorvalve. a Uaterr LI Bondapak C c o l u m (30 cm i 3.9 m, i.d.1, a Turner f l u o r m t e r . and a Hewlett-Packard 3385A P r i n t e r - p l b 8 t e r IUtC%atiOn System.
28. 29.
30. 31. 32.
33. 34.
35. 36. 31. 38.
39.
40. 41.
42. 43. 44. 45. 46.
U:
2'3: 51.
52.
..
1.
ml, ~
~
~
~
~
h
~
, 4,,32-103. H ~ " ~ . ~ d ~ ~ Protelnr, Pmno Adds, and Peptides: Reinhold
~
PublishingCmp.. New York, p. 375. Gibbons. 1. A. 1972. I n "Glycoproteins", E l r e v i e r , N e w York. P. 78. Lee. J . t . and Tlmarheff. 8 . N.. (1974).
A. b t t s c h a l k , ed.. 2nd ed.. P a r t
a,.
B i o c h e m l r t r ~ ~257-265. .
RESULTS
Amino t e r m n a l anal s i s : The amino terminal residue was determined by automated sequencer analysis (Beckkn W e 1 8% sequencer) using therrethcd of E h n and Begg (38) with dimthylbeniylamine (OWA) buffer and t h e '"general" program p l b l i r h e d by Henmdran e t ai. (39). Ten "J ofproteln(approximately 80 nrmlesl was suspended i n 500 "1 ofwater. NH OH placedintothespinning CUP. and dissolvedbyaddingthreedmprofconcentrated d r i e d w i t h t h e Beckman sample a p p l i c a t i o n r u b r o u t i & program. The sample was treatedwith sequencing by S t a r t i n g heptafluorobutyric acid (HFBA)andwashed with chlorobutane prior to t h e f i r s t sequencer c y c l e a t t h e HFBA a d d i t i o n Step (391. The p r o t e i n film UII sorewhat i n s o l u b l e i n WBA coupling buffer and w 3 therefore mbJected t o two cycles of coupling PliOP t O cleavage. Sequencer f r a c t i o n s e r e converted as p r e v i o v l l y r e p o r t e d (40) except that no ethaneth7ol was added t o t h e H C l , and thechlorobutanethat was used to extract the 3-phenyl. 2-thiohydantoin (PTC)-amino acid frMI the spinning cup contained dithiothreitol (15 mg/l). The r e s u l t i n g PTH sample was dissolved i n 20 y l of methanel. and 0.5 y l was examined on high Performance t h i n - l a y e r C h r m t o g r a p h i c p l a t e r ( E . Herck)coatedwith silica gel using the solventsystms Of Bucher 141). The remainder of the sample was d i l u t e d t o 50u1 w i t h using an SP-400 column methanol and a 1.5 "1 sample was analyzed by gas-liquid chrmtography as reportedpreviourly (401. The remaining PTH material was d i l u t e d t o 150 y l w t h methanol and analyzedbyhighperformance l i q u i d chromatography (HPLC). HPLC analyses were p e r f o m d w i t h a Varlan Model 5020 pump equipped w i t h a 20.~1 sample loop on a Rheodyne7120 i n j e c t i o n valve and a Tracor Hodel 970 UY-VIS v a r i a b l e wavelength d e t e c t o r (wavelength = 257 om). A OuPont 5-urn P d r t l c l e zorbax-008 column (25 M X 4.4 m) was used i n the reverse phase node a t 60°C.The detectorOutput was p l o t t e d and peaks were Integrated by a Hcwlett-Packard Hodel 3385A p r l n t e r - p l o t t e r . Sequencer r a w l e r were analyzed w i t h two d i f f e r e n t HPLC solvent System, one a gradient and One an isOCrdtlC method. The gradient inethad was a m a d i f l c a t i o n Of t h a t of Bhown e t 1 1 . (42) which involved Iubstituting acetonitrile for wthanol to reduce back pressurel and to increase mars transfereffects. The column #as deYeloped by s t a r t i n g w i t h a 9 mi" gradient of 95% a c e t o n i t r i l e ( A ) - 5 % sodlum n. pH 4 . 5 ( 8 ) t o 801A-2018 Iflowr a t e = 2.5 nlfmfn),operatingilOCmtiCally acetate,0.1 (70%A-30$8) f o r 9 m i " a t 1 rnllmin and inmediately returning to the i n i t i a lg r a d i e n t conditions. This procedure s l l a e d a complete ChrDmatOoraPhiC analvsis ~ Y C P Y20 mln. The
Table I .
LethalCMcentrationof subunit, and purified
H. IeXta was placed On thetreatedSurface of each contalner. The COnStdllt temperature (Z7'Cl and humidlty ( 5 0 % ) u n t i l cups containing the Iarvae-weFZGptat theObsewatlonperiod ended. A m o r t a l i t y C h l n t was made after 7 days Of exposure tothe treated ~ w f a c eand thelarvae w e m Characterizedsccordlngtostadia1development.Control larvae were incubated i n cups cantalninydietwithoutpurifiedmaterial. 111data were SubJected t o p w b i t a n a l y r i r ( 4 7 ) . was
pLIISPOPa1 L r y l t l l , a l k a l i - s o l u b i l i z e d
LethalConcentration 10-14
ng/c.' Parasporalcrystal
mle/cml
7.0 (5.5-8.5Ia
Protoxin
2.7 (1.4-3.4)
2.0 11.0-2.51
TWi"
1.1 (0.7-1.71
1.6 (1.0-2.51
'95%
confidence l i m i t s l n parentheses.
Amino acld
Residues per mol w t of 68,O0Oa ParaspordlCrystal Protoxin
Toxin
Lyrlne Histidine
16
IO
13
8
Argimne
48
46
Aspartic acid
77
80
37 98
Threonine
39
36
38
Seri"e
47
39
61
Glutamic72 acid P r o l i n e 32
73
69
21
Glycine
45
41
25 48
Alamne
15
12
45 33 10
32
IO
8
Valine MethlOnlneb
42
44
5
5
33 8
Iroleudne Leucine
34
33
30
48
45
48
Tyronne
25
23
Phenylalanine
24
29 6
21 31
TIYptOphdnC
Of
0. t h u r i n i e n s i r toTiin-sexta.
Preparatlo"
Half-cyrtlneb
Malewlarwelqhtdetermnation: The m o l e c ~ l d rwe?ght of solubilizedCrystalProtein determinedby agarore g e l f i l t r a t i o n ~n quanidlmum hydrachlande (24). vltracentrlfugation ( P a ) , and polyacrylamidegelelectraphorens ~n 80s ( 2 8 )
z,
212.
Carboh d m t e anal lis: Totalcarbohydrates were deteminedbytheanthronereaction 136). S l a l r c m d d e r t v a t i v e l were measured w i t h t h i o b a r b i t u r i c a d d a f t e r h y d m l y r i r i n 0.1N H$04 a t 80°C f a r 60 rnin (37). Amino sugars w e ~ emeasured in HCl hydrolysates (see a m n o a d d a n a l y r i r above) by ion-exchange chromatography on a Beckman120C amino acid analyzer.
One neonate l a r v a
PdPeTl
Bulla, k. A.. J r . , Bennett, G. A , . and S h o t w l l . 0. L. (1970). J . Bacterial. 1 0 4 , 1246-1253. Yeber K. Pringle. J. R . and Orbarn, M. (1972). I n 8 . P . C o l w l c k and N. 0. Kaplan (ed.): &hods i n Enzyolaqy, '401, 26, Part C. Acadmic Press In< New York, p. 3-27. Omstein, L. and Day>%. 8. 3 . (1962).Reprintedby D i s t i l l a t i o n Products i n d u l t T l e % , Rochester, N.Y. Arai A. and Wallace H. U. (1969). lnd. Biochm. 31 71-76. P. H. ( 1 9 k l . J . B l o l . Chem. 250. 4 0 0 7 - n i l . 0'Fl;rell Lowry. O.'H., Rorebraugh. N. J . . Farr, A . T . and Randall, R . J . (1951). J. 8101 Chem. 193 265-275. Hips, m r U . (1967) i n Hethodl in Enzymology ( C a l w i c k . 8 . P . and Kapldn. N. 0.. edr) '401. 11, pp. 59-62. Academic PPSI I n c . . New Yark. Y . H. (1971). J . B i o l . Chem. 246. 2842-2848. L i u T.-Y. and Chang Hili. 0. Y:, Walters.'F. H. yi1~011. T. 0.. and S t u a r t T . D. (1979).Anal. Chem. 1338-1341. Roe. J . H . (1955). J . B i o l . Chem. 335-313. Warren L. (1959). J . B i o l . Chem. 234, 1971-1975. Edman.'?., and Begg. G. (1967). E u T J . Biochem. 80-91 Hernadron. M.A.. Ericslon, 1. H . , Titmi, K.. Neurath. H.. and Ualrh, K . A. 11972). Biochem. 11, 4493-4502. Mak. A. S, and Jones, 8. L. (19761. Can. J . Biochm. 5 4 , 835-842. Bucher, 0. (1917). C h r m t o g r a p h i a E. 723-725. Bhwn, A. 8.. Hole, J. E., Ueirrlnger. A,. and Bennett. J . C . (1978). J . Chromat. 148 532-535. z&-n!an C. 1. (1977). Anal. B i o c h m . 77 569-573. Rmon, R.'11970). I n 8. P.Colaickand'N7b. Kap1a.n (ed.1. Pethods 7 " Enynaloqv. '401. 19.. Academic Press. In
