Int. J. Peptide Protein Rex 10, 1917, 252-264 Published by Munksgaard, Copenhagen, Denmark. No part may be reproduced by any process without written permission from the author(s)
I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F A NEW TRYPSIN-LIKE ENZYME FROM Tenebrio rnolitor L. L A R V A E HAIM LEVINSKY, YEHUDITH BIRK and SHALOM W. APPLEBAUM
Departments of Agricultural Biochemistry and Entomology, Faculty of Agriculture, The Hebrew University o f Jerusalem, Rehovot, Israel
Received 23 March, accepted for publication 1 April 1977 A trypsin-like enzyme (TLE) was separated and purified from Tenebrio molitor larval midgut enzyme solution by ion-exchange chromatography on a DEAEcellulose column. The purified enzyme was found to be a homogeneous protein by electrophoresis in polyacrylamide gels and on cellulose acetate strips, by electrofocusing in polyacrylamide gels and by SDS-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 18 300 by ultracentrifugal analysis and 24 300 on SDS-polyacrylamide gel electrophoresis. It has an isoelectric point 8.0, it contains only four half-cystine residues per molecule and the NH2-terminal amino acid is isoleucine. TLE possesses a high degree o f specificity towards trypsin synthetic substrates such as Na-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME) and poly-Llysine hydrobromide. The optimal pH f o r TLE activity was found to be 8.0 and the optimal temperature 50’C. Its Km value when assayed on BAPNA was 0.93mM and on TAME 0.08mM. TLE is stable at neutral pHs and its activity is not affected by Caz+ and by 0.01 M 1,4-dithiothreitol (DTT). I t is inactivated by DFP and tosyl-L-lysine chloromethylketone (TLCK) and is fully inhibited by the naturally occurring trypsin inhibitors such as trypsinand a-chymotrypsin inhibitor ( A A ) from soybeans, basic pancreatic trypsin inhibitor (BPTI), chick peas trypsin and chymotrypsin inhibitor (CI) and crystalline soybean trypsin inhibitor (CSBTI), forming with them complexes in a molar ratio o f 1 :I. The Ki value for A A with BAPNA as substrate is 5.87. lo-’ M and f o r BPTI 7.92 * 1O-’M. No common antigenic determinants were noted between TLE and bovine trypsin. This finding together with the relatively low number o f -S-S- bonds in the TLE molecule indicate that TLE differs in conformation from bovine trypsin.
I
Key words: insect trypsin-larval proteases-tenebrio proteases-tenebrio trypsin-tenebrio trypsin and trypsin inhibitors-tenebrio trypsin kinetics
Mammalian pancreatic trypsin (E.C. 3.4.4.4) is a well-known enzyme. It is present throughout the vertebrates from dogfish to humans (Reeck et aL, 1970; Hermodson et aZ., 1971). In recent years enzymes described as trypsins or trypsin-like were found in various micro252
organisms as well as in invertebrates (Keil, 1971). All of them belong to the category of the serine proteases, they reveal tryptiG cleavage specificity on synthetic and protein substrates, and are inhibited by naturally occurring as well as by synthetic trypsin in-
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
hibitors. Enzymes responding to the criteria established for trypsin have been demonstrated in the Actinomycetes Steptumyces griseus (pronase) (Trop & Birk, 1968) and Strepromyces fradiae (Morihara & Tsuzuki, 1968). The most extensive studies on insect trypsin have been made on cocoonase, the proteolytic enzyme produced by silk moths for softening the end of the cocoon to permit exit of the adult moth (Kafatos et al., 1967 a, b ) . Our studies have concentrated on the proteolytic enzymes of the digestive tract of Tenebrio mulitor larvae. The meal worm Tenebrio molitor lives in granaries and causes extensive damage there. Earlier attempts to study its larval midgut enzymes, have shown the presence of an endopeptidase “Tenebrio trypsin” with a considerable trypsin- like activity (Appelbaum et al., 1964). Later Zwilling et al. (1968; 1972) described a method for the purification of a-and 0-proteases from the digestive tract of adult Tenebrio molitor beetles. They pointed out that aprotease differs markedly from bovine trypsin and chymotrypsin and that the P-protease, which is inhibited by all natural trypsin inhibitors, lacks cleavage specificity on chain B of oxidized insulin. The trypsin-related serine proteases of animal and bacterial origin are examples of sequence-homologous proteins of divergent functions (Haen et al., 1975). Very little information is available in this respect on proteolytic enzymes from the digestive tract of insects. The present paper reports the isolation, purification and characterization of a trypsin- like enzyme from Tenebrio molitor larval midgut. The activity and catalytic properties of the enzyme are examined by specificity and inhibitor studies. EXPERIMENTAL PROCEDURES Material
DEAE- cellulose (DE32) was obtained from Whatman, England. p-Tosyl-L-arginine methyl ester (TAME), N-acetyl-L-tyrosine ethyl ester (ATEE), diisopropyl phosphorofluoridate (DFP), tosyl-L-lysin, chloromethylketone (TLCK), 1,4-dithiothreitoI (DTT), 5 3 dithiobis
(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoate (p-CMB), bovine serum albumin (BSA), and carboxypeptidase A and B were obtained from Sigma Chemical Co., U.S.A. Ampholine pH 3.5-10 was from LKB-Producter AB, Sweden. Trypsin twice crystallized, a-chymotrypsin three times crystallized, ovalbumin, crystalline soybean trypsin inhibitor (CSBTI) and lysozyme were products of Worthington Co., U.S.A. Poly-L-lysine hydrobromide was purchased from Miles-Yeda, Ltd., Israel. N - a-benzoyl-DL-arginine-p-nitroanilide (BAPNA) was a product of Merk, Germany. Chymotrypsinogen, casein and pnitrophenyl-p-guanidinobenzoate (p-NPGB) were purchased from Nutritional Biochemicals Corporation, U.S.A. Coomassi brilliant blue (R-250) from Serva, Germany. Amido black (12B), sodium dodecyl sulfate (SDS)and urea were obtained from British Drug Houses, Ltd., England. Dansylchloride and polyacrylamide were from Fluka AG, Switzerland. Chick pea trypsin and chymotrypsin inhibitor (CI) was isolated and purified according to Smirnoff et al. (1976). Trypsin- and a-chymotrypsininhibitor (AA) from soybeans (The BowmanBirk inhibitor) was isolated and purified according to Birk & Gertler (1968). Basic pancreatic trypsin inhibitor (BPTI), a product of Laboratoire Choay, France, was a gift from Dr. M. Rigbi, Hebrew University, Jerusalem, Israel. Polyamide t.1.c. plates were obtained from Cheng Chin Trading Co. Ltd., Formoza. Enzyme assays
Proteolytic activity was determined at pH 7.6 by the casein digestion method of Kunitz (1947) as described by Laskowski (1955), measuring the change in absorbancy at A:E. Trypsin and chymotrypsin activities were determined titrimetrically in a Radiometer pH stat model TTT 1 equipped with pH meter 26, with Recorder SBRZ, and with a syringe burette type SBUl in which 0.1 N NaOH was used as titrant. The reactions were carried out in 0.003M tris-4.045M KCl 0.01 5 M CaClz 2H20 buffer, pH 8.0, at 30’ C by using p-tosyl-Larginine methyl ester (TAME) 0.015M- as the substrate for trypsin 253
H. LEVINSKY. Y. BIRK and S.W.APPLEBAUM
and N-acetyl-L-tyrosine ethyl ester (ATEE) (Dixon et al., 1958) and tosyl-L-lysine 0.019M - as the substrate for chymotrypsin chloromethyl ketone (TLCK) (Shaw et al., (Neurath & Schwert, 1950). One unit of 1965; Shaw & Glover, 1970) was studied in enzymatic activity was equal to the hydro- a series of experiments with TAME as sublysis of 1 pmol substrate/min. The specific strate. Enzymatic activity was measured after activity was defined as units of activity per preincubation of the enzyme with the inmg of enzyme protein. Spectrophotometric hibitor at various concentrations and for assay of trypsin activity was carried out on different lengths of time and the time rethe substrate 0.05 M N-cu-benzoyl-DL-arginine- quired for 50% inhibition was calculated. Active-site titration was carried out with p-nitroanilide (BAPNA) in 0.14 M tris 0.025 M p-nitrophenyl p-guanidinobenzoate HCl (jCaC12.2H20 buffer pH 8.0 at 30°C by measuring the change in absorbancy at accord- N E B ) in a Gilford 2400 spectrophotometer ing to Erlanger et al. (1961). Trypsin speci- at 410nm in 0.1 M sodium veronal - 0.02M ficity was also studied chromatographically CaC12 buffer pH 8.3 (Chase & Shaw, 1970). on poly-L-lysine hydrobromide as described The effect of 0.02M Ca2+ on TLE was exby Katchalski et al. (1961) and Waley & Wat- amined with TAME as substrate. The effects son (1953). Carboxypeptidase B activity was of 4 M urea and 0.01 M DTT were examined determined according to Folk et al. (1960) on BMNA as substrate. with several modifications. The substrate 0.006 M hippuryl-L-arginine (2 mglml) was reacted with enzyme samples dissolved in Preparation of the trypsin-like enzyme (TLE) 0.025M tris-O.l M NaCl pH 7.6. Samples were withdrawn from the reaction mixture Tenebrio molitor larvae were grown at 24°Cafter different time intervals, applied on Silica 26°C on a diet containing 90% wheat bran and gel G plates and chromatographed in a solvent 10% flaked oats. Larvae (-130 mg each) were system composed of 75 g phenol + 25 g water. dissected behind the thoracic region and the Arginine spots were detected by spraying the midgut was taken out and cleared of body fat. plates with a solution of 0.3% ninhydrin in Midguts, at a concentration of three per 1 ml 100 ml n-butanol containing 1.5 ml glacial of cold distilled water, were homogenized in’ acetic acid. Protein content was determined a chilled tissue grinder with teflon pestle. The by the method of Lowry et al. (1951) and homogenate was centrifuged in a Sorvall model expressed in absorbancy units. Emergence of RC-2B refrigerated superspeed centrifuge at protein solution from the chromatographic 17300xg for 20min. The supernatant, desigcolumns was monitored by measuring the nated midgut enzyme solution (MES), was absorbancy at 280 nm. Carbohydrate con- lyophilized and stored at -20°C (yielding tent was determined by the anthrone method about 3 mg lyophilized MES per one midgut). according to Scott & Melvin (1953) and by A sample of 1300mg lyophilized MES was phenol-sulfuric acid according to Dubois et al. dissolved in 15 ml of 0.001 M ammonium (1956). For detection of glycoproteins the acetate buffer pH 6.5 (the starting buffer) SDS-polyacrylamide gel electrophoresis method and applied to two DEAE- cellulose (DE-32) columns (2 x 58 cm) that had been previously was used (Segest & Jackson, 1972). equilibrated with 0.001 M ammonium acetat$ buffer pH 6.5. Elution, at room temperature, Effect of inhibitors, active site titrants and was performed primarily with 0.001 M ammonium acetate buffer pH 6.5. The flow rate was reagents adjusted to 80 ml/h and 3 ml-fractions were The effect of naturally occurring trypsin collected. Each tube was assayed for protein inhibitors was determined as described by content by measuring the absorbancy at 289 Birk et al. (1963). nm, for tryptic activity on TAME and for The effects of the active-site directed in- chymotryptic activity on ATEE. The tubes hibitors diisopropyl phosphorfluoridate (DFP) containing trypsin activity only (TLE) were
&fr
254
TRYPSIN-LIKEENZYME FROM Tenebrio molitor
combined, dialyzed against distilled water with constant stirring at 4°C (eight changes, every hour against 100 vol. of distilled water each) and stored at -20°C (henceforth, dialyzed TLE). For certain studies, dialyzed TLE was freeze-dried and stored in a desicator. For further purification, TLE was freeze-dried immediately after emergence from the DEAEcellulose column, dissolved in a minimal volume (about 2ml) of 0.001 M ammonium acetate buffer pH 6.5 and rechromatographed on a DEAE-cellulose column (2 x 58cm) using the same conditions as above. The effluent was collected, dialyzed, freeze-dried and stored in a desiccator.
according to Ouchterlony (1953), by the double diffusion method in 1% (W/V) agarose gel in 0.9% NaCl layered thinly on glass slides (7 x 7cm). Amino acid and end group analyses
For amino acid analyses 0.5mg samples of freeze-dried TLE were dissolved in 1 ml 6N HC1 and hydrolyzed in evacuated tubes for 24h at 110°C in the presence of a known amount of DL-norleucine as standard. The hydrolyzed samples were analyzed by a single I
1
I
2.1
1
1.4
Criteria for purity
Disc electrophoresis analyses were performed in 7.5% polyacrylamide gel at pH 4.5, with [%7.5 mA/tube for 60min. (Reisfeld etal., 1962). Electrophoresis on cellulose acetate strips was performed in a Beckman Microzone electrophoresis cell model R-100 in 0.08M collidine acetate buffer pH 7.3. One-pl samples of 1 md25 pl protein solution were applied to the strip and electrophoresis was performed at a constant voltage of 400V for 20min. The strips were stained with 0.2% amido black (1 2B) in methanol/Hz O/acetic acid (45 :45 : 10 by vol.). Isoelectrofocusing was performed by the method of Wrigley (1968) in 7.5% polyacrylamide gels with a pH gradient in the range of 3.5-10. For reference, one gel was electro'focused without protein, and the pH gradient in this gel was checked by measuring the pH of slices of the gel soaked in distilled water. SDS- polyacrylamide gel electrophoresis was performed on solutions of protein samples which were incubated at 37OC for 2 h in 0.01 M sodium phosphate buffer pH 7.0 containing 1% SDS and 1% 0-mercaptoethanol. The samples were applied to gels by the procedure of Weber & Osborn (1969). Molecular weight determinations were performed by the highspeed equilibrium method of Yphantis (1960) in a Spinco model E ultra%centrifuge,using a partial specific volume ($ resolved from the amino acid composition. Immunodiffusion studies were performed
1.2
1.c
0.0
:
80.6 0.4
0.2
30
\IUMBER
40
FIGURE 1 Chromatography of 650mg lyophilized MES on a DEAEcellulose column (2 X 50 cm)equilibrated with 0.001 M ammonium acetate buffer pH 6.5 and eluted with the same buffer. (-), protein absorbancy at 280nm; (A-), trypsin-like activity on TAME; (0-o), chymotrypsin-like activity on ATEE.
255
H . LEVINSKY, Y. BIRK and S.W.APPLEBAUM TABLE 1 hrification of a trypsin-like enzyme (TLE) from Tenebrio molitor larval midgut enzyme solution (MES) Fraction
MES TLE Dialyzed TLE Rechromatographed TLE'
Vol. (ml)
A,*,,,
Absorbance units
15.0 15.9
166.00 0.720
2490.00 11.45
15.9
0.515
17.2
0.312
SpeCific activity (units/mg pro tein)
Total activity (units)
Yield
(%I
Purification (fold)
4.3 546.8
4442 2591
100 58.3
1 127.1
8.19
734.3
2498
56.2
170.7
5.36
741.8
1650
37.1
172.5
* For Anal removal of accompanying carbohydrates from the DEAE-cellulose columns it is sometimes necessary to precipitate with (NH,), SO, (100% saturation).
column separation program in a LKB 3201 amino acid analyzer. Cysteic acid was determined after performic acid oxidation according to Hirs (1967). Tryptophan was determined after 24 h hydrolysis in the presence of 4% thioglycollic acid (Matsubara & Sasaki, 1969). The NH2-terminal amino acid was determined by the dansyl-chloride method on polyamide sheets (Rosmus & Deyl, 1971). The COOH-terminal amino acid was determined with carboxypeptidase B and carboxypeptidase A (Fraenkel-Conrat et al., 1955). Sulfhydryl content was determined with p-chloromercuribenzoate (p-CMB) according to Benesh & Benesh (1962) and with Ellman's (1959) reagents 5.5' dithiolbis (2-nitrobenzoic acid) (DTNB) as described by Schram (1964). RESULTS
together and either lyophilized or rechromatographed for final purification. The preparation procedure of TLE is summarized in Table 1.) Since it has been found that dialysis of TLE against distilled water at 4°C results in the same specific activity achieved by rechromatography of TLE and with almost no loss of total activity, the dialyzed TLE was used for the activity assays. Rechromatographed TLE was used for analytical purposes only. The electrophoretic pattern of TLE on polyacrylamide gels at pH 4.5 showed that theenzyme behaved as a pure homogenous band migrating towards the cathode. Similar results were obtained by isoelectric focusing in polyacrylamide gels and by SDS - polyacrylamide gel electrophoresis (Fig. 2a, b). An isoelectric pH of 8.0 was resolved from the isoelectricfocusing analysis. It was found that Coomassie brilliant blue (R-250) stains TLE samples better than amido black (12B).
Purification and homogeneity
Preliminary assays of MES on specific synthetic substrates indicated the presence of trypsin-, chymotrypsin- and carboxypeptidase B-like enzymes. The separation pattern of a sample of MES on a DEAE-cellulose column is shown in Fig. 1. Two active peaks emerged with the hold-up volume. The first peak (tube nos. 20-30) contained mainly chymotrypsin-like activity, whereas the second peak (tube nos. 32-38) showed only trypsin-like activity. Tubes 33-35 of the TLE peak were pooled 256
Characterization
Most of the experiments were performed withL TLE in comparison with bovine trypsin (henceforth trypsin). TLE has a specific extinction coefficient of A:% = 24.1 at 280nm. (NH,), S04-precipitated TLE has been found to be free of carbohydrates, when examined by the anthron or phenol methods. Specific staining for glycoproteins of ~ d samples analyzed by SDS-polyacrylamide gel electrophoresis was found to be negative. The
'
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
1’
1
2
3
FIGURE 2a SDS-acrylamide gel electrophoresis of rechromatographed TLE (40 fig), dialyzed TLE (40 fig) and bovine trypsin (50rg). The enzymes were incubated in 0.01 M phosphate buffer pH 7.0 containing 1% SDS, 1% j7-mercapto1 applied to the gel. ethanol and 5 pl of 0.05% bromphenol blue in water for 2 h at 37°C. Samples of 5 0 ~ were Electrophoresis was performed in 10% acrylamide gels using 0.1 M phosphate buffer pH 7.0 containing 0.1% SDS at a constant current of 8 mA per tube for 3 h. Staining was achieved using Coomassie brilliant blue (R-250) (Weber & Osborn, 1969). (1) Bovine trypsin. (2) Rechromatographed TLE. (3) Dialyzed TLE. FIGURE 2b Jsoelectricfocusing of TLE in 7.5% acrylamide gels in a pH gradient of pH 3.5-10 from top to bottom. A sample of 100 fig TLE was applied per tube and the isoelectric focusing was performed for 5 h at 350V with maximal current of 1 mA per tube. Staining was achieved with Coomassie brilliant blue (R 250) (Chrombach e l af.,1967).
molecular weight of TLE, as calculated from ultracentrifugal analysis using a partial specific volume (3 of 0.72, is 18300. A value of 2 4 300 is obtained by the SDS-polyacrylamide gel electrophoresis (Fig. 3). For specific activity calculations a value of 20 000 was used. The amino acid composition of TLE as compared with that of trypsin (Walsh & Neurath, 1964), pronase trypsin (Jurasek et al., 4969) and cocoonase (Kafatos et al., 1967a), is given in Table 2. It can be seen that the amino acid composition of TLE is similar to those of pronase trypsin and cocoonase. It
should, however, be pointed out that TLE lacks tryptophan and contains only four halfcystine residues per molecule of protein, when measured as cysteic acid after performic acid oxidation. No free sulfhydryl groups were found in TLE with either of the reagents (pCMB or DTNB) employed. The NH2-terminal amino acid is isoleucine, and the COOH terminal amino acid is asparagine or threonine. Specificity
The action of TLE on the synthetic substrate 257
H. LEVINSKY, Y. BIRK and S.W. APPLEBAUM TABLE 2 Amino acid composition and N-terminal amino acid of TLE, bovine rrypsin, cocoonase and Streptomyces griseus (pronase) trypsin Amino acid
TLE
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Halfcystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
3 3 6 16 13 33 13 8 30 16 4 19 1 12 13 8 1 None
Total N-terminal amino acid
199 Ile
‘
.JL -
I
Bovine trypsin (Walsh & Neurath, 1964)
Cocoonase (Kafatos et al., 1967a)
14 3 2 22 10 33 14 9 25 14 12 17 2 15 14 10 3 4
13 4 6 26 16 23 15 9 22 16 4 20 1-2 12 12 9 5 3
223 Ile (Haen etal., 1975)
I
1
I
216-217 Ile (Haenat al.. 1975)
‘
b -
-
I-
-
x -
2 W 3 3-
-
CK
43 V
w, 0
I 0
258
4
-
25-
1-
6.5 1.o 8.4 18.1 16.4 14.2 17.4 8.0 28.4 26.0 6.1 17.8 2.7 8.0 11.0 8.2 5.1 Not determined 203.9 Val (Haen et al., 1975)
-
Bovine serum
- 7-
U
Pronase trypsin (Jurasek et al., 1969)
0.4 0.8 RELATIVE MOB I LlTY
1.2
FIGURE 3 Estimation of the mol.wt. of rechromatographed TLE by SDS-gel electrophoresis. Gels and samples were prepared as indicated by Weber & Osborn (1969). Bovine serum albumin (68 OOO), ovalbumin (43 000), chymotrypsinogen (25 700), crystallin soybean trypsin i~ hibitor (21 500) and lysozyme (14300) were used as molecular size markers.
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
LYS
LYS3
LYS
a 1
2
3
i
-4
4
6
5
7
a
FIGURE 4 Hydrolysis of poly-L-lysine by TLE or trypsin. The reaction mixture was composed of 22mg/ml of poly-Llysine hydrobromide in a solution of 0.04M phosphate buffer pH 7.6 containing 0.083M KCI and 12pg TLE or lOpg trypsin. Samples of 20pl were applied on Whatman No. 3 paper. A descending chromatogram was Sun for 48 h at room temperature using acetic acidln-butanol/pyridine/water (6 :30 :24 :30 by vol.) as solvent. Spots were detected with 0.3% (w/v) ninhydrin in n-butanol containing 3% (v/v) acetic acid glacial. (1) Trypsin. (2) TLE. (3) Poly-L-lysine. (4) Trypsin + poly-L-lysine. ( 5 ) TLE lx poly-L-lysine. (6) TLE 2x + poly-L-lysine. (7) [TLE + AA] + poly-L-lysine. The complex between TLE and trypsh- and cxchymotrypsininhibitor (AA) from soybean was formed in 1 :1 molar ratio. ( 8 ) TLE AA.
+
-
+
poly-l-lysine hydrobromide is shown in Fig. of TLE and trypsin, with casein and BAPNA 3. It is obvious that TLE acts in a similar as substrates, was examined in the pH ranges manner to trypsin, yielding free lysine, dilysine, trilysine and tetralysine. TLE also hydrolyzes readily the synthetic substrates BAPNA and TAME in a similar manner to trypsin, as will be shown in the following paragraph.
of 5.1-8.4 and of 3.4-9.4, respectively. The optimal pH for TLE on both substrates was found to be 8.0 and for trypsin 7.8. The effect of temperature on activity was studied in the range of 2O"C-7O0C. Optimal proteolytic and esterolytic activities were obtained at 50°C for TLE and at 5OoC-55"C Kinetic properties for trvtxin. Tiirkion of the enzyme with p-NPGB at The comparative effect of pH on the activities 259
H. LEVINSKY, Y. BIRK and S.W.APPLEBAUM 5 (0)
L
2
- 2 ~ -20
1
-16
-12
-8
-4
[$] x 104 M
[+] 103 M FIGURE 5 Lineweaver-Burk plots of TLE ( 0 ) and trypsin (a) against various concentrations of (a) BAPNA and (b) TAME. (a) v = A,,, m/15 min per reaction mixture. (b) v = pmol/min per reaction mixture.
pH 8.3 and subsequent calculation of the concentration of active enzyme from the observed burst of p-nitrophenol at 410nm revealed 78% of active enzyme. The effect of substrate concentration was studied with the substrates BAPNA and TAME. The Km and Kcat values for TLE were calculated from the Lineweaver-Burk plot (Line-
(a) Semilogarithmic inactivation plots of TLE (0.9 * lo-' M) and trypsin (1.0*10-' M) by two different concentrations of DFP (1.0*10'3M and 2.5.10-'M) at room temperature in 0.05M tris buffer pH 8.0 containing 0.02M calcium ion, when assayed on TAME as substrate. (b) Semilogarithmic inactivation plots of TLE (0.9.10-'M) and trypsin (1.0. 10*M) by l.lO"M TLCK at room temperature in 0.05 M tris buffer p~ 8.0 containing 0.02 M calcium ion, when assayed on TAME as substrate. ( A ) , trypsin residual activity; (o---o), TLE residual activity.
260
weaver & Burk, 1934) (Fig. 5a, b) as following: the Km on BAPNA was 0.93 mM and on TAME 0.08mM and the Kcat values were 0.548 sec-' and 170 sec-' ,respectively. Stability
TLE was dissolved in water or in 0.001 M HCl and kept for 5 0 h at 4°C and -20°C. TLE at 4"C, both in water and in HCl retained its full activity on casein and -80% of its activity on BMNA. Storage at -20°C for 50h had no effect on either activities of TLE when it was dissolved in water, but resulted in almost a complete loss of both activities when TLE was stored in 0.001 M HCI. The comparative effect of Ca2+ on the activities of TLE and trypsin was examined in enzyme solutions in 0.05M tris-HC1 pH 8.0, which were allowed to stand for 3.5h at 37°C and for 48 h at 24°C in the presence and absence of 0.02 M CaC12 -2Hz 0 and then' assayed on TAME at pH 8.0. TLE retained its full activity in the absence of Ca2+, whereas trypsin was inactivated rapidly under the same experimental conditions (Green & Neurath, 1953). Examination of the effects of urea and D n on TLE and trypsin when assayed on BAPNA revealed that 4 M urea in reaction mixture caused 60% inactivation of T U and 90% inactivation of t,.,,psin. on fie other hand, 0.01 M DTT in reaction mixture did not affect TLE at all, but almost fully inhibited trypsin. When the enzymes were assayed in the presence of both 4 M urea and 0.001 M DTT, the extent of inactivation of TLE did not extend its 60% while trypsin was fully inactivated.
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
Effects of inhibitors
TLE and trypsin, respectively, and for BPTI 7.92.10-7M and 5.42-10-'M on these enzymes The comparative effects of DFP and TLCK on TLE and trypsin when assayed on TAME as respectively. Both inhibitors behave as competitive inhibitors. substrate are presented in Fig. 6a, b. Both TLE readily forms complexes with the enzymes are inactivated by DFP at about the naturally occurring trypsin inhibitors, such same rate. The half-life calculated at concenas CSBTI, AA and CI. The complexes formed trations of l ~ l o - ~ M DFP and 2.5.10-4M between TLE and CSBTI as well as between DFP was around 2.5 min and 8.5 min, respecttrypsin and CSBTI are shown by cellulose ively, for both enzymes. However, TLE was acetate electrophoresis in 0.08 M collidine inactivated faster by TLCK than trypsin, the acetate buffer pH 7.3 (Fig. 8). half-life calculated at 1 M TLCK was 3.8 min for TLE and 8.7 min for trypsin. Antigenicity The effect of naturally occurring trypsin inhibitors: soybean trypsin and crchymotrypsin No cross-reaction was noted between TLE inhibitor (AA) and basic pancreatic trypsin and antibodies to bovine trypsin even at inhibitor (BPTI), when assayed on casein and relatively high concentrations of TLE (Fig. BAPNA as substrates, is shown in Fig. 7a, b. 9A, B, C) indicating that TLE and trypsin The Ki values, as calculated from the Line- lack common antigenic determinants. weaver-Burk plots with BAPNA as substrate 'are for AA 5.87.10-7M and 5.07.10-7M on DISCUSSION
1
LI
0 0
L1-d 21 L 1. 2 1.
6 6
0 0
2 2
1. 1.
6 6
8 8
p g INHIRITOR IN 1 ml REACTION MIXTURE
10 10
12 12
FIGURE 7 7a) Inhibition of the activity of TLE and trypsin on BAPNA by naturally occurring trypsin inhibitors. 1 ml reaction mixture contained 3.25 pg TLE or 5.35 wg trypsin and various amounts of the inhibitors BPTI and AA. (b) Inhibition of the proteolytic activity of TLE and trypsin on casein by naturally occurring trypsin inhibitors. 1 ml reaction mixture contained 17.42pg TLE or 3.12pg trypsin and various amounts of the inhibitors BPTI and AA. ( a d ) , trypsin residual activity; (0-0), TLE residual activity.
The proteinases of insects are of interest from the points of view of comparative biochemistry and enzyme evolution. Considerable studies have been devoted to proteinases of insects that infest stored products. A major objective of these studies was to provide basic information for the possible utilization of naturally occurring inhibitors of proteolysis, present in stored-product seeds and grains, as built-in mechanisms of resistance against the attack of insects (Applebaum & Birk, 1972). Most of the presently known trypsin-related serine proteases originate from the digestive system of vertebrates and from microorganisms. The homology in their sequences was found to extend into the activation peptide. In the present work we have isolated and characterized a trypsin-like enzyme from the larval midgut of Tenebrio molitor. TLE fully resembles bovine trypsin with respect to size, substrate specificity, kinetic parameters, inhibitability by synthetic- and naturally occurring trypsin inhibitors and active-site titrants. It is rather unlikely that TLE would differ markedly in its primary structure from the other trypsinlike serine proteases. A few characteristics suggest, however, possible conformational differences between TLE and bovine trypsin. Both enzymes are inactivated by DFP at the 26 1
H. LEVINSKY, Y. BIRK and S.W. APPLEBAUM
FIGURE 8 Cellulose acetate electrophore sis analyses of TLE and twp sin and their complexes witha naturally occurring trypsinin hibitor. The electrophoresis was performed at room tem perature in 0.08M collidine acetate buffer pH 7.3at constant voltage of 4001V TLE and trypsin weremixed with CSBTI at different in hibitor/enzyme molar ratios (1) CSBTI. (2) Trypsin.(3) CSBTI + Trypsin (1 : 1 molar ratio). (4) TLE. ( 5 ) CSBTI+ TLE (1 :0.4 molar ratio).(6) TLE. (7) CSBTI + TLE (1:2) molar ratio). (8) CSBTI.
'
FIGURE 9 Double immunodiffusion of TLE and trypsh onagar The diffusion was performed with 0.1 ml of enzyme solution in the following concentrations,againt 0.15 ml of antitrypsin. (A) 1.2mg TLE (treated or not treated withDFP) in 1 ml saline and 3 mg trypsin (treated or nottreated with DFP) in 1 ml saline. (B) TLE and trypsin in I X and 3x concentrations as in (A). (C) 3.5mgTLE (DFP-treated) in 1 ml saline and 9 mg trypsh (DFP treated) in 1 ml saline.
262
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
same rate, but TLE is inactivated faster by TLECK than trypsin (Fig. 6a, b) indicating the possibility that the equivalent of His 57 in TLE is more accessible to the reagent than His 57 in trypsin. In addition, TLE possesses only ofur half-cystines (as compared to the six disulfide bridges in trypsin) which a priori pxescribe no more than two disulfide bonds per molecule. Moreover, treatment of TLE and trypsii with excess of dithiothreitol, which resulted in full inactivation of the trypsin, did notaffect at all the activity of TLE. Unfolding ofthemolecule by 4M urea did not seem to facilitate the access of the reducing agent to the presumably hidden -S-S-bondsin TLE. the net-charge differences between TLE and trypsin, expressed in their different electrophoretic mobility (Fig. 8) and in amino acid composition (Table 2), as well as the lack of requirement for CaZ+ for the stabilization of TLE should be pointed out as well. So far Tenebrio molitor larvae seem to be the only example of a stored-product pest to possess trypsin, chymotrypsin and carboxypeptidase B in thei digestive tract. It is suggested that they may serve as a suitable model for selective impairment of the development of stored-product pests by naturally occurring proteinase inhibitors such as those present in llgume seeds and cereal grains (Birk, 1976). ACKNOWLEDGEMENT
k i s study was supported by Grant No. 161 from the United States-Israel Binational Science Foundation
W) REFERENCES Applebaum, S.W., Birk, Y., Harpaz, I. & Bondi, A. 1964) Comp. Biochem. Physiol. 11,85-103 Applebaum, S.W. & Birk, Y. (1972) inInsect andMite Nutrition (Rodriguez, J.G., ed.), pp. 629-636, North-Holland,Amsterdam Benesh, R. & Benesh, R.E. (1962) Methods Biochem. Anal 10,43-70 Bi1k.Y 11976) in Methods in Enzymology (Lorand, ( L., d), ~ 0 1 - XLV [56 - 631 pp. 695-739, Academic Press, New York Bi1k.Y ., Gertler, A. & Khalef, S. (1963) Biochem. J. 87., 281-284 Bi1k.Y. & Gertler, A. (1968) Biochem. Prep. 12, 25 - 29
Chase, T., Jr. & Shaw, E. (1970) in Methods in Enzymology (Perlman, G.E. & Lorand, L., eds.), vol. XIX, pp. 20-27, Academic Press, New York Chrombach, A., Reisfield, R.A., Wyckoff, M. & Zaccari, J. (1967) Anal. Biochem. 20,150-154 Dixon, G.H., Kauffman, D.L. & Neurath, H. (1958) J. Biol. Chem. 233,1373-1381 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. (1956) Anal. Chem. 28, 350-356 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77 Erlanger, B.F., Kokowsky, N. & Cohen, W. (1961) Arch. Biochem. Biophys. 95,271-278 Folk, J.E., Piez, K.A., Carrol, W.R. & Gladner, J.A. (1960) J. Biol. Chem. 235,2272-2277 FraenkelConrat, H., Harris, J.I. & Levy, A.L. (1955) Methods Biochem. Analy. 2,359-425 Green, N.M. & Neurath, H. (1953) J. Biol. Chem. 204,379-390 Haan, D.C., Neurath, H. & Teller, D.C. (1975) J. Mol. Biol. 92,225-259 Hermodson, M.A., Tye, R.W., Reeck, G.R., Neurath, H. & Walsh, K.A. (1971) FEBS Letters 14, 222-224 Hirs, C.H.W. (1967) in Methods in Enzymology (Hirs, C.H.W., ed.), vol. XI, pp. 59-62, Academic Press, New York Jurasek, L., Fackne, D. & Smillie, L.B. (1969) Biochem. Biophys. Res. Commun. 37,99-105 Kafatos, F.C., Tartakoff, A.M. & Law, J.H. ( 1 9 6 7 ~ ) J. Bwl. Orem. 242,1477-1487 Kafatos, F.C., Law, J.H. & Tartakoff, A.M. (1967b) J Biol. Chem. 242, 1477-1487 Katchalski, E., Levin, Y., Neumann, H., Riesel, E. & Sharon, N. (1961) Bull. Rex Council Israel 10A, 159-171 Keil, B. (1971) in The Enzymes (Boyer, P.D., ed.), vol. 111, pp. 249-275, Academic Press, New York Kunitz, M. (1947) J. Gen. Physiol. 30,291-310 Laskowski, M. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.), vol. 11, pp. 8-36, Academic Press, New York Lineweaver, H. & Burk, D.J. (1934) Am. Chem. SOC.56,658-666 Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951) J. Biol. Chem. 193,265-275 Matsubara, H. & Sasaki, R.M. (1969) Biochem. Biophys. Res. Commun. 35,175-181 Morihara, K. & Tsuzuki, H. (1968) Arch. Biochem. Biophys. 126,971-973 Neurath, H. & Schwert, G.W. (1950) Chem. Rev. 46,69-153 Ouchterlony, 0. (1953) A c f a Pathol. Microbiol. Scand. 32,231-240
263
H. LEVINSKY. Y. BIRK and S.W. APPLEBAUM Reeck, G.R., Winter, W.P. & Neurath, H. (1970) Biochemistry 9,1398-1403 Reisfield, R.A., Lewis, U.J. & Williams, D.E. (1962) Nature (Lond.) 195,281-283 Rosmus, J. & Deyl, Z. (1971) Chromatog. Rev. 13, 16 3- 302 Schram, M. (1964) Biochemistry 3,1231-1234 Scott, T.A., Jr. & Melvin, E.H. (1953) AML Chem 25, 1656-1661 Segrest, J.P. & Jackson, R.L. (1972) in Methods in Enzymology (Ginsburg, V., ed.). vol. XXVIII, Part B, pp. 54-63 Academic Press, New York Shaw, E., MaresGuia, M. & Cohen, W. (1965) Biochemistry 4,2219-2224 Shaw, E. & Glover, G. (1970) Arch. Biochem. Biophys. 139,298-305 Smirnoff, P., Khalef, S., Birk, Y. & Applebaum, S.W. (1976) Biochem. J. 157,745-751 Trop, M. & Birk, Y. (1968) Biochem. J. 109,475-476
264
Waley, S.G. & Watson, J. (1953) Biochem. J. 55, 328-336 Walsh, K.A. & Neurath, H. (1964) Proc. Natl. Acad. Sci. U.S.A. 52,884-889 Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 Wrigley, C . (1968) Sci. Tools 15,17-23 Yphantis, D.A. (1960) Ann. N.Y. Acad. Sci. 88, 586-601 Zwilling, R. (1968) Hoppe-SeylerS 2. Physiol. Chem. 349,326-332 Zwilling, R., Medugorac, 1. & Mella, K. (1972) Comp. Biochem. Physiol. 43B,419-424 Address: Prof. Y. Birk c/o Hormone Research Laboratory University of California San Francisco, CA 94143 U.S.A.
I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F A NEW TRYPSIN-LIKE ENZYME FROM Tenebrio rnolitor L. L A R V A E HAIM LEVINSKY, YEHUDITH BIRK and SHALOM W. APPLEBAUM
Departments of Agricultural Biochemistry and Entomology, Faculty of Agriculture, The Hebrew University o f Jerusalem, Rehovot, Israel
Received 23 March, accepted for publication 1 April 1977 A trypsin-like enzyme (TLE) was separated and purified from Tenebrio molitor larval midgut enzyme solution by ion-exchange chromatography on a DEAEcellulose column. The purified enzyme was found to be a homogeneous protein by electrophoresis in polyacrylamide gels and on cellulose acetate strips, by electrofocusing in polyacrylamide gels and by SDS-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 18 300 by ultracentrifugal analysis and 24 300 on SDS-polyacrylamide gel electrophoresis. It has an isoelectric point 8.0, it contains only four half-cystine residues per molecule and the NH2-terminal amino acid is isoleucine. TLE possesses a high degree o f specificity towards trypsin synthetic substrates such as Na-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME) and poly-Llysine hydrobromide. The optimal pH f o r TLE activity was found to be 8.0 and the optimal temperature 50’C. Its Km value when assayed on BAPNA was 0.93mM and on TAME 0.08mM. TLE is stable at neutral pHs and its activity is not affected by Caz+ and by 0.01 M 1,4-dithiothreitol (DTT). I t is inactivated by DFP and tosyl-L-lysine chloromethylketone (TLCK) and is fully inhibited by the naturally occurring trypsin inhibitors such as trypsinand a-chymotrypsin inhibitor ( A A ) from soybeans, basic pancreatic trypsin inhibitor (BPTI), chick peas trypsin and chymotrypsin inhibitor (CI) and crystalline soybean trypsin inhibitor (CSBTI), forming with them complexes in a molar ratio o f 1 :I. The Ki value for A A with BAPNA as substrate is 5.87. lo-’ M and f o r BPTI 7.92 * 1O-’M. No common antigenic determinants were noted between TLE and bovine trypsin. This finding together with the relatively low number o f -S-S- bonds in the TLE molecule indicate that TLE differs in conformation from bovine trypsin.
I
Key words: insect trypsin-larval proteases-tenebrio proteases-tenebrio trypsin-tenebrio trypsin and trypsin inhibitors-tenebrio trypsin kinetics
Mammalian pancreatic trypsin (E.C. 3.4.4.4) is a well-known enzyme. It is present throughout the vertebrates from dogfish to humans (Reeck et aL, 1970; Hermodson et aZ., 1971). In recent years enzymes described as trypsins or trypsin-like were found in various micro252
organisms as well as in invertebrates (Keil, 1971). All of them belong to the category of the serine proteases, they reveal tryptiG cleavage specificity on synthetic and protein substrates, and are inhibited by naturally occurring as well as by synthetic trypsin in-
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
hibitors. Enzymes responding to the criteria established for trypsin have been demonstrated in the Actinomycetes Steptumyces griseus (pronase) (Trop & Birk, 1968) and Strepromyces fradiae (Morihara & Tsuzuki, 1968). The most extensive studies on insect trypsin have been made on cocoonase, the proteolytic enzyme produced by silk moths for softening the end of the cocoon to permit exit of the adult moth (Kafatos et al., 1967 a, b ) . Our studies have concentrated on the proteolytic enzymes of the digestive tract of Tenebrio mulitor larvae. The meal worm Tenebrio molitor lives in granaries and causes extensive damage there. Earlier attempts to study its larval midgut enzymes, have shown the presence of an endopeptidase “Tenebrio trypsin” with a considerable trypsin- like activity (Appelbaum et al., 1964). Later Zwilling et al. (1968; 1972) described a method for the purification of a-and 0-proteases from the digestive tract of adult Tenebrio molitor beetles. They pointed out that aprotease differs markedly from bovine trypsin and chymotrypsin and that the P-protease, which is inhibited by all natural trypsin inhibitors, lacks cleavage specificity on chain B of oxidized insulin. The trypsin-related serine proteases of animal and bacterial origin are examples of sequence-homologous proteins of divergent functions (Haen et al., 1975). Very little information is available in this respect on proteolytic enzymes from the digestive tract of insects. The present paper reports the isolation, purification and characterization of a trypsin- like enzyme from Tenebrio molitor larval midgut. The activity and catalytic properties of the enzyme are examined by specificity and inhibitor studies. EXPERIMENTAL PROCEDURES Material
DEAE- cellulose (DE32) was obtained from Whatman, England. p-Tosyl-L-arginine methyl ester (TAME), N-acetyl-L-tyrosine ethyl ester (ATEE), diisopropyl phosphorofluoridate (DFP), tosyl-L-lysin, chloromethylketone (TLCK), 1,4-dithiothreitoI (DTT), 5 3 dithiobis
(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoate (p-CMB), bovine serum albumin (BSA), and carboxypeptidase A and B were obtained from Sigma Chemical Co., U.S.A. Ampholine pH 3.5-10 was from LKB-Producter AB, Sweden. Trypsin twice crystallized, a-chymotrypsin three times crystallized, ovalbumin, crystalline soybean trypsin inhibitor (CSBTI) and lysozyme were products of Worthington Co., U.S.A. Poly-L-lysine hydrobromide was purchased from Miles-Yeda, Ltd., Israel. N - a-benzoyl-DL-arginine-p-nitroanilide (BAPNA) was a product of Merk, Germany. Chymotrypsinogen, casein and pnitrophenyl-p-guanidinobenzoate (p-NPGB) were purchased from Nutritional Biochemicals Corporation, U.S.A. Coomassi brilliant blue (R-250) from Serva, Germany. Amido black (12B), sodium dodecyl sulfate (SDS)and urea were obtained from British Drug Houses, Ltd., England. Dansylchloride and polyacrylamide were from Fluka AG, Switzerland. Chick pea trypsin and chymotrypsin inhibitor (CI) was isolated and purified according to Smirnoff et al. (1976). Trypsin- and a-chymotrypsininhibitor (AA) from soybeans (The BowmanBirk inhibitor) was isolated and purified according to Birk & Gertler (1968). Basic pancreatic trypsin inhibitor (BPTI), a product of Laboratoire Choay, France, was a gift from Dr. M. Rigbi, Hebrew University, Jerusalem, Israel. Polyamide t.1.c. plates were obtained from Cheng Chin Trading Co. Ltd., Formoza. Enzyme assays
Proteolytic activity was determined at pH 7.6 by the casein digestion method of Kunitz (1947) as described by Laskowski (1955), measuring the change in absorbancy at A:E. Trypsin and chymotrypsin activities were determined titrimetrically in a Radiometer pH stat model TTT 1 equipped with pH meter 26, with Recorder SBRZ, and with a syringe burette type SBUl in which 0.1 N NaOH was used as titrant. The reactions were carried out in 0.003M tris-4.045M KCl 0.01 5 M CaClz 2H20 buffer, pH 8.0, at 30’ C by using p-tosyl-Larginine methyl ester (TAME) 0.015M- as the substrate for trypsin 253
H. LEVINSKY. Y. BIRK and S.W.APPLEBAUM
and N-acetyl-L-tyrosine ethyl ester (ATEE) (Dixon et al., 1958) and tosyl-L-lysine 0.019M - as the substrate for chymotrypsin chloromethyl ketone (TLCK) (Shaw et al., (Neurath & Schwert, 1950). One unit of 1965; Shaw & Glover, 1970) was studied in enzymatic activity was equal to the hydro- a series of experiments with TAME as sublysis of 1 pmol substrate/min. The specific strate. Enzymatic activity was measured after activity was defined as units of activity per preincubation of the enzyme with the inmg of enzyme protein. Spectrophotometric hibitor at various concentrations and for assay of trypsin activity was carried out on different lengths of time and the time rethe substrate 0.05 M N-cu-benzoyl-DL-arginine- quired for 50% inhibition was calculated. Active-site titration was carried out with p-nitroanilide (BAPNA) in 0.14 M tris 0.025 M p-nitrophenyl p-guanidinobenzoate HCl (jCaC12.2H20 buffer pH 8.0 at 30°C by measuring the change in absorbancy at accord- N E B ) in a Gilford 2400 spectrophotometer ing to Erlanger et al. (1961). Trypsin speci- at 410nm in 0.1 M sodium veronal - 0.02M ficity was also studied chromatographically CaC12 buffer pH 8.3 (Chase & Shaw, 1970). on poly-L-lysine hydrobromide as described The effect of 0.02M Ca2+ on TLE was exby Katchalski et al. (1961) and Waley & Wat- amined with TAME as substrate. The effects son (1953). Carboxypeptidase B activity was of 4 M urea and 0.01 M DTT were examined determined according to Folk et al. (1960) on BMNA as substrate. with several modifications. The substrate 0.006 M hippuryl-L-arginine (2 mglml) was reacted with enzyme samples dissolved in Preparation of the trypsin-like enzyme (TLE) 0.025M tris-O.l M NaCl pH 7.6. Samples were withdrawn from the reaction mixture Tenebrio molitor larvae were grown at 24°Cafter different time intervals, applied on Silica 26°C on a diet containing 90% wheat bran and gel G plates and chromatographed in a solvent 10% flaked oats. Larvae (-130 mg each) were system composed of 75 g phenol + 25 g water. dissected behind the thoracic region and the Arginine spots were detected by spraying the midgut was taken out and cleared of body fat. plates with a solution of 0.3% ninhydrin in Midguts, at a concentration of three per 1 ml 100 ml n-butanol containing 1.5 ml glacial of cold distilled water, were homogenized in’ acetic acid. Protein content was determined a chilled tissue grinder with teflon pestle. The by the method of Lowry et al. (1951) and homogenate was centrifuged in a Sorvall model expressed in absorbancy units. Emergence of RC-2B refrigerated superspeed centrifuge at protein solution from the chromatographic 17300xg for 20min. The supernatant, desigcolumns was monitored by measuring the nated midgut enzyme solution (MES), was absorbancy at 280 nm. Carbohydrate con- lyophilized and stored at -20°C (yielding tent was determined by the anthrone method about 3 mg lyophilized MES per one midgut). according to Scott & Melvin (1953) and by A sample of 1300mg lyophilized MES was phenol-sulfuric acid according to Dubois et al. dissolved in 15 ml of 0.001 M ammonium (1956). For detection of glycoproteins the acetate buffer pH 6.5 (the starting buffer) SDS-polyacrylamide gel electrophoresis method and applied to two DEAE- cellulose (DE-32) columns (2 x 58 cm) that had been previously was used (Segest & Jackson, 1972). equilibrated with 0.001 M ammonium acetat$ buffer pH 6.5. Elution, at room temperature, Effect of inhibitors, active site titrants and was performed primarily with 0.001 M ammonium acetate buffer pH 6.5. The flow rate was reagents adjusted to 80 ml/h and 3 ml-fractions were The effect of naturally occurring trypsin collected. Each tube was assayed for protein inhibitors was determined as described by content by measuring the absorbancy at 289 Birk et al. (1963). nm, for tryptic activity on TAME and for The effects of the active-site directed in- chymotryptic activity on ATEE. The tubes hibitors diisopropyl phosphorfluoridate (DFP) containing trypsin activity only (TLE) were
&fr
254
TRYPSIN-LIKEENZYME FROM Tenebrio molitor
combined, dialyzed against distilled water with constant stirring at 4°C (eight changes, every hour against 100 vol. of distilled water each) and stored at -20°C (henceforth, dialyzed TLE). For certain studies, dialyzed TLE was freeze-dried and stored in a desicator. For further purification, TLE was freeze-dried immediately after emergence from the DEAEcellulose column, dissolved in a minimal volume (about 2ml) of 0.001 M ammonium acetate buffer pH 6.5 and rechromatographed on a DEAE-cellulose column (2 x 58cm) using the same conditions as above. The effluent was collected, dialyzed, freeze-dried and stored in a desiccator.
according to Ouchterlony (1953), by the double diffusion method in 1% (W/V) agarose gel in 0.9% NaCl layered thinly on glass slides (7 x 7cm). Amino acid and end group analyses
For amino acid analyses 0.5mg samples of freeze-dried TLE were dissolved in 1 ml 6N HC1 and hydrolyzed in evacuated tubes for 24h at 110°C in the presence of a known amount of DL-norleucine as standard. The hydrolyzed samples were analyzed by a single I
1
I
2.1
1
1.4
Criteria for purity
Disc electrophoresis analyses were performed in 7.5% polyacrylamide gel at pH 4.5, with [%7.5 mA/tube for 60min. (Reisfeld etal., 1962). Electrophoresis on cellulose acetate strips was performed in a Beckman Microzone electrophoresis cell model R-100 in 0.08M collidine acetate buffer pH 7.3. One-pl samples of 1 md25 pl protein solution were applied to the strip and electrophoresis was performed at a constant voltage of 400V for 20min. The strips were stained with 0.2% amido black (1 2B) in methanol/Hz O/acetic acid (45 :45 : 10 by vol.). Isoelectrofocusing was performed by the method of Wrigley (1968) in 7.5% polyacrylamide gels with a pH gradient in the range of 3.5-10. For reference, one gel was electro'focused without protein, and the pH gradient in this gel was checked by measuring the pH of slices of the gel soaked in distilled water. SDS- polyacrylamide gel electrophoresis was performed on solutions of protein samples which were incubated at 37OC for 2 h in 0.01 M sodium phosphate buffer pH 7.0 containing 1% SDS and 1% 0-mercaptoethanol. The samples were applied to gels by the procedure of Weber & Osborn (1969). Molecular weight determinations were performed by the highspeed equilibrium method of Yphantis (1960) in a Spinco model E ultra%centrifuge,using a partial specific volume ($ resolved from the amino acid composition. Immunodiffusion studies were performed
1.2
1.c
0.0
:
80.6 0.4
0.2
30
\IUMBER
40
FIGURE 1 Chromatography of 650mg lyophilized MES on a DEAEcellulose column (2 X 50 cm)equilibrated with 0.001 M ammonium acetate buffer pH 6.5 and eluted with the same buffer. (-), protein absorbancy at 280nm; (A-), trypsin-like activity on TAME; (0-o), chymotrypsin-like activity on ATEE.
255
H . LEVINSKY, Y. BIRK and S.W.APPLEBAUM TABLE 1 hrification of a trypsin-like enzyme (TLE) from Tenebrio molitor larval midgut enzyme solution (MES) Fraction
MES TLE Dialyzed TLE Rechromatographed TLE'
Vol. (ml)
A,*,,,
Absorbance units
15.0 15.9
166.00 0.720
2490.00 11.45
15.9
0.515
17.2
0.312
SpeCific activity (units/mg pro tein)
Total activity (units)
Yield
(%I
Purification (fold)
4.3 546.8
4442 2591
100 58.3
1 127.1
8.19
734.3
2498
56.2
170.7
5.36
741.8
1650
37.1
172.5
* For Anal removal of accompanying carbohydrates from the DEAE-cellulose columns it is sometimes necessary to precipitate with (NH,), SO, (100% saturation).
column separation program in a LKB 3201 amino acid analyzer. Cysteic acid was determined after performic acid oxidation according to Hirs (1967). Tryptophan was determined after 24 h hydrolysis in the presence of 4% thioglycollic acid (Matsubara & Sasaki, 1969). The NH2-terminal amino acid was determined by the dansyl-chloride method on polyamide sheets (Rosmus & Deyl, 1971). The COOH-terminal amino acid was determined with carboxypeptidase B and carboxypeptidase A (Fraenkel-Conrat et al., 1955). Sulfhydryl content was determined with p-chloromercuribenzoate (p-CMB) according to Benesh & Benesh (1962) and with Ellman's (1959) reagents 5.5' dithiolbis (2-nitrobenzoic acid) (DTNB) as described by Schram (1964). RESULTS
together and either lyophilized or rechromatographed for final purification. The preparation procedure of TLE is summarized in Table 1.) Since it has been found that dialysis of TLE against distilled water at 4°C results in the same specific activity achieved by rechromatography of TLE and with almost no loss of total activity, the dialyzed TLE was used for the activity assays. Rechromatographed TLE was used for analytical purposes only. The electrophoretic pattern of TLE on polyacrylamide gels at pH 4.5 showed that theenzyme behaved as a pure homogenous band migrating towards the cathode. Similar results were obtained by isoelectric focusing in polyacrylamide gels and by SDS - polyacrylamide gel electrophoresis (Fig. 2a, b). An isoelectric pH of 8.0 was resolved from the isoelectricfocusing analysis. It was found that Coomassie brilliant blue (R-250) stains TLE samples better than amido black (12B).
Purification and homogeneity
Preliminary assays of MES on specific synthetic substrates indicated the presence of trypsin-, chymotrypsin- and carboxypeptidase B-like enzymes. The separation pattern of a sample of MES on a DEAE-cellulose column is shown in Fig. 1. Two active peaks emerged with the hold-up volume. The first peak (tube nos. 20-30) contained mainly chymotrypsin-like activity, whereas the second peak (tube nos. 32-38) showed only trypsin-like activity. Tubes 33-35 of the TLE peak were pooled 256
Characterization
Most of the experiments were performed withL TLE in comparison with bovine trypsin (henceforth trypsin). TLE has a specific extinction coefficient of A:% = 24.1 at 280nm. (NH,), S04-precipitated TLE has been found to be free of carbohydrates, when examined by the anthron or phenol methods. Specific staining for glycoproteins of ~ d samples analyzed by SDS-polyacrylamide gel electrophoresis was found to be negative. The
'
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
1’
1
2
3
FIGURE 2a SDS-acrylamide gel electrophoresis of rechromatographed TLE (40 fig), dialyzed TLE (40 fig) and bovine trypsin (50rg). The enzymes were incubated in 0.01 M phosphate buffer pH 7.0 containing 1% SDS, 1% j7-mercapto1 applied to the gel. ethanol and 5 pl of 0.05% bromphenol blue in water for 2 h at 37°C. Samples of 5 0 ~ were Electrophoresis was performed in 10% acrylamide gels using 0.1 M phosphate buffer pH 7.0 containing 0.1% SDS at a constant current of 8 mA per tube for 3 h. Staining was achieved using Coomassie brilliant blue (R-250) (Weber & Osborn, 1969). (1) Bovine trypsin. (2) Rechromatographed TLE. (3) Dialyzed TLE. FIGURE 2b Jsoelectricfocusing of TLE in 7.5% acrylamide gels in a pH gradient of pH 3.5-10 from top to bottom. A sample of 100 fig TLE was applied per tube and the isoelectric focusing was performed for 5 h at 350V with maximal current of 1 mA per tube. Staining was achieved with Coomassie brilliant blue (R 250) (Chrombach e l af.,1967).
molecular weight of TLE, as calculated from ultracentrifugal analysis using a partial specific volume (3 of 0.72, is 18300. A value of 2 4 300 is obtained by the SDS-polyacrylamide gel electrophoresis (Fig. 3). For specific activity calculations a value of 20 000 was used. The amino acid composition of TLE as compared with that of trypsin (Walsh & Neurath, 1964), pronase trypsin (Jurasek et al., 4969) and cocoonase (Kafatos et al., 1967a), is given in Table 2. It can be seen that the amino acid composition of TLE is similar to those of pronase trypsin and cocoonase. It
should, however, be pointed out that TLE lacks tryptophan and contains only four halfcystine residues per molecule of protein, when measured as cysteic acid after performic acid oxidation. No free sulfhydryl groups were found in TLE with either of the reagents (pCMB or DTNB) employed. The NH2-terminal amino acid is isoleucine, and the COOH terminal amino acid is asparagine or threonine. Specificity
The action of TLE on the synthetic substrate 257
H. LEVINSKY, Y. BIRK and S.W. APPLEBAUM TABLE 2 Amino acid composition and N-terminal amino acid of TLE, bovine rrypsin, cocoonase and Streptomyces griseus (pronase) trypsin Amino acid
TLE
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Halfcystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
3 3 6 16 13 33 13 8 30 16 4 19 1 12 13 8 1 None
Total N-terminal amino acid
199 Ile
‘
.JL -
I
Bovine trypsin (Walsh & Neurath, 1964)
Cocoonase (Kafatos et al., 1967a)
14 3 2 22 10 33 14 9 25 14 12 17 2 15 14 10 3 4
13 4 6 26 16 23 15 9 22 16 4 20 1-2 12 12 9 5 3
223 Ile (Haen etal., 1975)
I
1
I
216-217 Ile (Haenat al.. 1975)
‘
b -
-
I-
-
x -
2 W 3 3-
-
CK
43 V
w, 0
I 0
258
4
-
25-
1-
6.5 1.o 8.4 18.1 16.4 14.2 17.4 8.0 28.4 26.0 6.1 17.8 2.7 8.0 11.0 8.2 5.1 Not determined 203.9 Val (Haen et al., 1975)
-
Bovine serum
- 7-
U
Pronase trypsin (Jurasek et al., 1969)
0.4 0.8 RELATIVE MOB I LlTY
1.2
FIGURE 3 Estimation of the mol.wt. of rechromatographed TLE by SDS-gel electrophoresis. Gels and samples were prepared as indicated by Weber & Osborn (1969). Bovine serum albumin (68 OOO), ovalbumin (43 000), chymotrypsinogen (25 700), crystallin soybean trypsin i~ hibitor (21 500) and lysozyme (14300) were used as molecular size markers.
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
LYS
LYS3
LYS
a 1
2
3
i
-4
4
6
5
7
a
FIGURE 4 Hydrolysis of poly-L-lysine by TLE or trypsin. The reaction mixture was composed of 22mg/ml of poly-Llysine hydrobromide in a solution of 0.04M phosphate buffer pH 7.6 containing 0.083M KCI and 12pg TLE or lOpg trypsin. Samples of 20pl were applied on Whatman No. 3 paper. A descending chromatogram was Sun for 48 h at room temperature using acetic acidln-butanol/pyridine/water (6 :30 :24 :30 by vol.) as solvent. Spots were detected with 0.3% (w/v) ninhydrin in n-butanol containing 3% (v/v) acetic acid glacial. (1) Trypsin. (2) TLE. (3) Poly-L-lysine. (4) Trypsin + poly-L-lysine. ( 5 ) TLE lx poly-L-lysine. (6) TLE 2x + poly-L-lysine. (7) [TLE + AA] + poly-L-lysine. The complex between TLE and trypsh- and cxchymotrypsininhibitor (AA) from soybean was formed in 1 :1 molar ratio. ( 8 ) TLE AA.
+
-
+
poly-l-lysine hydrobromide is shown in Fig. of TLE and trypsin, with casein and BAPNA 3. It is obvious that TLE acts in a similar as substrates, was examined in the pH ranges manner to trypsin, yielding free lysine, dilysine, trilysine and tetralysine. TLE also hydrolyzes readily the synthetic substrates BAPNA and TAME in a similar manner to trypsin, as will be shown in the following paragraph.
of 5.1-8.4 and of 3.4-9.4, respectively. The optimal pH for TLE on both substrates was found to be 8.0 and for trypsin 7.8. The effect of temperature on activity was studied in the range of 2O"C-7O0C. Optimal proteolytic and esterolytic activities were obtained at 50°C for TLE and at 5OoC-55"C Kinetic properties for trvtxin. Tiirkion of the enzyme with p-NPGB at The comparative effect of pH on the activities 259
H. LEVINSKY, Y. BIRK and S.W.APPLEBAUM 5 (0)
L
2
- 2 ~ -20
1
-16
-12
-8
-4
[$] x 104 M
[+] 103 M FIGURE 5 Lineweaver-Burk plots of TLE ( 0 ) and trypsin (a) against various concentrations of (a) BAPNA and (b) TAME. (a) v = A,,, m/15 min per reaction mixture. (b) v = pmol/min per reaction mixture.
pH 8.3 and subsequent calculation of the concentration of active enzyme from the observed burst of p-nitrophenol at 410nm revealed 78% of active enzyme. The effect of substrate concentration was studied with the substrates BAPNA and TAME. The Km and Kcat values for TLE were calculated from the Lineweaver-Burk plot (Line-
(a) Semilogarithmic inactivation plots of TLE (0.9 * lo-' M) and trypsin (1.0*10-' M) by two different concentrations of DFP (1.0*10'3M and 2.5.10-'M) at room temperature in 0.05M tris buffer pH 8.0 containing 0.02M calcium ion, when assayed on TAME as substrate. (b) Semilogarithmic inactivation plots of TLE (0.9.10-'M) and trypsin (1.0. 10*M) by l.lO"M TLCK at room temperature in 0.05 M tris buffer p~ 8.0 containing 0.02 M calcium ion, when assayed on TAME as substrate. ( A ) , trypsin residual activity; (o---o), TLE residual activity.
260
weaver & Burk, 1934) (Fig. 5a, b) as following: the Km on BAPNA was 0.93 mM and on TAME 0.08mM and the Kcat values were 0.548 sec-' and 170 sec-' ,respectively. Stability
TLE was dissolved in water or in 0.001 M HCl and kept for 5 0 h at 4°C and -20°C. TLE at 4"C, both in water and in HCl retained its full activity on casein and -80% of its activity on BMNA. Storage at -20°C for 50h had no effect on either activities of TLE when it was dissolved in water, but resulted in almost a complete loss of both activities when TLE was stored in 0.001 M HCI. The comparative effect of Ca2+ on the activities of TLE and trypsin was examined in enzyme solutions in 0.05M tris-HC1 pH 8.0, which were allowed to stand for 3.5h at 37°C and for 48 h at 24°C in the presence and absence of 0.02 M CaC12 -2Hz 0 and then' assayed on TAME at pH 8.0. TLE retained its full activity in the absence of Ca2+, whereas trypsin was inactivated rapidly under the same experimental conditions (Green & Neurath, 1953). Examination of the effects of urea and D n on TLE and trypsin when assayed on BAPNA revealed that 4 M urea in reaction mixture caused 60% inactivation of T U and 90% inactivation of t,.,,psin. on fie other hand, 0.01 M DTT in reaction mixture did not affect TLE at all, but almost fully inhibited trypsin. When the enzymes were assayed in the presence of both 4 M urea and 0.001 M DTT, the extent of inactivation of TLE did not extend its 60% while trypsin was fully inactivated.
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
Effects of inhibitors
TLE and trypsin, respectively, and for BPTI 7.92.10-7M and 5.42-10-'M on these enzymes The comparative effects of DFP and TLCK on TLE and trypsin when assayed on TAME as respectively. Both inhibitors behave as competitive inhibitors. substrate are presented in Fig. 6a, b. Both TLE readily forms complexes with the enzymes are inactivated by DFP at about the naturally occurring trypsin inhibitors, such same rate. The half-life calculated at concenas CSBTI, AA and CI. The complexes formed trations of l ~ l o - ~ M DFP and 2.5.10-4M between TLE and CSBTI as well as between DFP was around 2.5 min and 8.5 min, respecttrypsin and CSBTI are shown by cellulose ively, for both enzymes. However, TLE was acetate electrophoresis in 0.08 M collidine inactivated faster by TLCK than trypsin, the acetate buffer pH 7.3 (Fig. 8). half-life calculated at 1 M TLCK was 3.8 min for TLE and 8.7 min for trypsin. Antigenicity The effect of naturally occurring trypsin inhibitors: soybean trypsin and crchymotrypsin No cross-reaction was noted between TLE inhibitor (AA) and basic pancreatic trypsin and antibodies to bovine trypsin even at inhibitor (BPTI), when assayed on casein and relatively high concentrations of TLE (Fig. BAPNA as substrates, is shown in Fig. 7a, b. 9A, B, C) indicating that TLE and trypsin The Ki values, as calculated from the Line- lack common antigenic determinants. weaver-Burk plots with BAPNA as substrate 'are for AA 5.87.10-7M and 5.07.10-7M on DISCUSSION
1
LI
0 0
L1-d 21 L 1. 2 1.
6 6
0 0
2 2
1. 1.
6 6
8 8
p g INHIRITOR IN 1 ml REACTION MIXTURE
10 10
12 12
FIGURE 7 7a) Inhibition of the activity of TLE and trypsin on BAPNA by naturally occurring trypsin inhibitors. 1 ml reaction mixture contained 3.25 pg TLE or 5.35 wg trypsin and various amounts of the inhibitors BPTI and AA. (b) Inhibition of the proteolytic activity of TLE and trypsin on casein by naturally occurring trypsin inhibitors. 1 ml reaction mixture contained 17.42pg TLE or 3.12pg trypsin and various amounts of the inhibitors BPTI and AA. ( a d ) , trypsin residual activity; (0-0), TLE residual activity.
The proteinases of insects are of interest from the points of view of comparative biochemistry and enzyme evolution. Considerable studies have been devoted to proteinases of insects that infest stored products. A major objective of these studies was to provide basic information for the possible utilization of naturally occurring inhibitors of proteolysis, present in stored-product seeds and grains, as built-in mechanisms of resistance against the attack of insects (Applebaum & Birk, 1972). Most of the presently known trypsin-related serine proteases originate from the digestive system of vertebrates and from microorganisms. The homology in their sequences was found to extend into the activation peptide. In the present work we have isolated and characterized a trypsin-like enzyme from the larval midgut of Tenebrio molitor. TLE fully resembles bovine trypsin with respect to size, substrate specificity, kinetic parameters, inhibitability by synthetic- and naturally occurring trypsin inhibitors and active-site titrants. It is rather unlikely that TLE would differ markedly in its primary structure from the other trypsinlike serine proteases. A few characteristics suggest, however, possible conformational differences between TLE and bovine trypsin. Both enzymes are inactivated by DFP at the 26 1
H. LEVINSKY, Y. BIRK and S.W. APPLEBAUM
FIGURE 8 Cellulose acetate electrophore sis analyses of TLE and twp sin and their complexes witha naturally occurring trypsinin hibitor. The electrophoresis was performed at room tem perature in 0.08M collidine acetate buffer pH 7.3at constant voltage of 4001V TLE and trypsin weremixed with CSBTI at different in hibitor/enzyme molar ratios (1) CSBTI. (2) Trypsin.(3) CSBTI + Trypsin (1 : 1 molar ratio). (4) TLE. ( 5 ) CSBTI+ TLE (1 :0.4 molar ratio).(6) TLE. (7) CSBTI + TLE (1:2) molar ratio). (8) CSBTI.
'
FIGURE 9 Double immunodiffusion of TLE and trypsh onagar The diffusion was performed with 0.1 ml of enzyme solution in the following concentrations,againt 0.15 ml of antitrypsin. (A) 1.2mg TLE (treated or not treated withDFP) in 1 ml saline and 3 mg trypsin (treated or nottreated with DFP) in 1 ml saline. (B) TLE and trypsin in I X and 3x concentrations as in (A). (C) 3.5mgTLE (DFP-treated) in 1 ml saline and 9 mg trypsh (DFP treated) in 1 ml saline.
262
TRYPSIN-LIKE ENZYME FROM Tenebrio molitor
same rate, but TLE is inactivated faster by TLECK than trypsin (Fig. 6a, b) indicating the possibility that the equivalent of His 57 in TLE is more accessible to the reagent than His 57 in trypsin. In addition, TLE possesses only ofur half-cystines (as compared to the six disulfide bridges in trypsin) which a priori pxescribe no more than two disulfide bonds per molecule. Moreover, treatment of TLE and trypsii with excess of dithiothreitol, which resulted in full inactivation of the trypsin, did notaffect at all the activity of TLE. Unfolding ofthemolecule by 4M urea did not seem to facilitate the access of the reducing agent to the presumably hidden -S-S-bondsin TLE. the net-charge differences between TLE and trypsin, expressed in their different electrophoretic mobility (Fig. 8) and in amino acid composition (Table 2), as well as the lack of requirement for CaZ+ for the stabilization of TLE should be pointed out as well. So far Tenebrio molitor larvae seem to be the only example of a stored-product pest to possess trypsin, chymotrypsin and carboxypeptidase B in thei digestive tract. It is suggested that they may serve as a suitable model for selective impairment of the development of stored-product pests by naturally occurring proteinase inhibitors such as those present in llgume seeds and cereal grains (Birk, 1976). ACKNOWLEDGEMENT
k i s study was supported by Grant No. 161 from the United States-Israel Binational Science Foundation
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264
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