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Aminopeptidase from the harvester termite Trinervitermes trinervoides (Sjöstedt): Distribution, purification, physical and chemical properties
Insect Biochem., Vol. 11, No. 3, pp. 311-321, 1981. Printed in Great Britain.
0020-1790/81/030311-11502.00/0 © 1981. Pergamon Press Ltd.
AMINOPEPTIDASE FROM THE HARVESTER TERMITE TRINER VITERMES TRINER VOIDES (SJOSTEDT): DISTRIBUTION, PURIFICATION, PHYSICAL AND CHEMICAL PROPERTIES M. C. VAN DER WESTHUIZEN , P. H. HEWITT a n d P. J. DU TOIT* Department of Zoology and Entomology and *Department of Biochemistry, University of the Orange Free State, Bloemfontein, Republic of South Africa (Received 17 April 1980: revised 11 November 1980) Abstract--Approximately 70~o of the digestive aminopeptidase of the harvester termite Trinervitermes trinervoides (Sj6stedt) is found in the midgut. The enzyme was purified using conventional methods. The diffusion coefficient, Stokesradiusandmolecularmassweredeterminedas5.58 x 10-Tcm2/sec, 3.8 x 10 -7 cm and 88,000 daltons respectively. The chemical properties were as follows: pH optimum between 8 and 9, preferred substrate leucine-p-nitroanilide, K m for leu-p-nitroanilide and lys-p-nitroanilide hydrolysis at pH 8.0 were 4.000 x 10-~ M and 2.048 x 10-~ M respectively, Ea, Q l0, AH and AS were 66.79, 2.59 and 64.24 kJ and 232.16 J/°K/mol respectively. Key Word Index: Harvester termite, Trinervitermes trinervoides, aminopeptidase purification, physical and chemical properties
INTRODUCTION THE PRESENCE o f a m i n o p e p t i d a s e has been d e m o n s t r a t e d in several insects (CHAPMAN, 1971; GILMOUR, 1961 a n d WIGGLESWORTH, 1965) but to o u r knowledge n o comprehensive study of any insect a m i n o p e p t i d a s e has yet been undertaken. This l a b o r a t o r y has reported on the properties of cellulase, trehalase a n d aryl-fl-glucosidase from Trinervitermes trinervoides. This p a p e r represents a further c o n t r i b u t i o n to o u r knowledge of digestive enzymes of this termite a n d of insects in general. MATERIALS AND METHODS
The total assay volume was 1.5 ml of 0.02 M Tris-HCl buffer, pH 8.5 containing 3 mM lys-p-nitroanilide and 100/A of enzyme preparation which was added last. Enzyme activity, expressed in/~mole p-nitroanilide released/min/ml, was calculated from the reaction rate during the initial 10 sec. The extinction coefficient of p-nitroanilide used in these calculations was 9.62 x 103 M -1 cm -~ (BARMAN, 1969). The hydrolysis of other amino acid p-nitroanilides was assayed under identical conditions. Ala-fl-napthylamide hydrolysis was determined by the method of LEE et al. (1971) and that of N-carbobenzoxy L-tyrosine-p-nitrophenylester (ZTNE) by the method of BARMAN(1969). Casein hydrolysis was determined by the method of SPins (1957). Distribution of aminopeptidase
Termites Workers and larvae were collected from a mound situated in the vicinity of Bloemfontain. Chemicals Sephadex products were supplied by Pharmacia Chemicals, Sweden. Alcohol dehydrogenase and kiesegel 60F254 plates were obtained from Merck, bovine serum albumin and RNAse from Seravac, SDS molecular weight markers and cytochrome c from BDH and ovalbumien from Miles Biochemicals. All other chemicals, which were supplied by Merck Chemicals, British Drug Houses and ICN Pharmaceuticals, were of analytical grade. Protein determination The spectrophotometric method of Warburg and Christian (LAYNE, 1957), standardised against 2~o (w/v) bovine serum albumin, was used. Enzyme assays Unless otherwise stated all assays were conducted at 30°C. All buffers were prepared according to GOMORI (1955). Aminopeptidase activity was assayed by following the release ofp-nitroanilide, at 405 nm, from lys-p-nitroanilide. 311
The digestive tracts of ten pre-cooled workers (4°C) were removed and cut into the following easily recognisable regions: foregut, midgut, mixed segment, hindgut 1 and hindgut 2 and 3. Mixing of the contents of the various regions was avoided as far as possible. The pooled regions were homogenized with a Potter-Elvejehm homogenizer in 5 ml of 0.03 M Tris-HC1 buffer, pH 8.5. This crude homogenate was centrifuged at 20,000 g for 30 min at 4°C and the supernatant filtered through glass wool to remove fatty material. Purification of aminopeptidase (a) Crude Extract (step I). T. trinervoides workers and larvae (125 g) were homogenized with a Waring blender in 600 ml of ice-cold 0.9~o NaCl solution for 10 min at maximum speed and thereafter for a further l0 min with a high speed top-drive homogenizer. More NaCl (0.9~o) was added to give a total volume of 1.25 I. (10 ml/g wet termites). After stirring at 4°C for 8 hr the slurry was filtered through eight thicknesses of pre-wetted cheese-cloth. The homogenate was centrifuged at 20,000 g for 45 min at 4°C and the supernatant filtered through glass wool to remove the surface lipid layer. After dialysing the supernatant against two changes of 25 I. distilled water for 8 hr, the protein concentration and the enzymatic activity were determined.
312
M . C . VAN DER WESTHUIZEN, P. H. HEWITT AND P. J. DU TOIl
(b) (NH4)2SO 4 fractionation (step 2). The crude supernatant was brought to 30~o saturation with solid (NH,)2SO 4 (DI JESO, 1968). After complete addition of (NH4)2SO 4 stirring was continued for 2 hr. The precipitate was removed by centrifugation at 20,000 g for 45 min at 4°C. The supernatant was filtered through glass wool and dialyzed against five changes of 25 I. distilled water before freeze drying. ( c) DEA E-cellulose chromatography (step 3). The freezedried protein from step 2 was dissolved in 0.02 m Tris-HCl buffer, pH 8.15, and dialyzed for 8 hr against two changes 21. each) of the same buffer. The protein solution was applied to a DEAE-cellulose (DE-52) column (2.2 cm i.d. × 60 cm) equilibrated with the same buffer. Protein was eluted with a linear NaCI gradient (0.0--0.4 M NaCI in 11.) at a flow rate of 21 ml/hr and collected in 10-11 ml fractions. Aminopeptidase containing fractions, (36-56), were combined, dialyzed against three changes (3 1. each) of distilled water for 12 hr and freeze-dried. (d) DEAE-cellulose chromatography (step 4). The protein obtained in step 3 was dialyzed as described in step 3 and then re-chromatographed on a DE-52 column (2.2 cm i.d. x 53 cm) using a 0--0.25 M NaCI gradient in 0.02 M Tris-HCl buffer, pH 8.15. Fractions 45-68 were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. (e) DEAE-Sephadex chromatography (step 5). The step 4 protein was dissolved and dialyzed as described in step 3 and then chromatographed on D E A E - S e p h a d e x A-25 column (1.9 cm i.d. × 28 cm) at a flow rate of 21 ml buffer (0.02 M Tris-HCl, pH 8.15) per hr. Fractions 36---64 were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. (f ) Sephadex G-200 chromatography (step 6). The protein from step 5 was dissolved in 0.15 M KCI solution, dialyzed for 8 hr against 2 L of this solution and fractionated on a 2.2 cm i.d. × 90 cm KCl-equilibrated column at a flow rate of 21 ml/hr. Fractions of 10 ml were collected. Fractions 56-74 were combined, dialyzed against 4 1. distilled water for 8 hr, and then lyophilized. The dried protein was dissolved and dialyzed against 0.016 citrate-phosphate buffer, pH 6.4. (g) CM-Sephadex chromatography (step 7). The dialysate was applied to a 2.2 cm i.d. x 60 cm CM-Sephadex C-25 column equilbrated in the same buffer. The column was developed at a flow rate of 21 ml/hr and 5-6 ml fractions were collected. The aminopeptidase-containi,Lg fractions (8-16) were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. Gel electrophoresis (pH 8.6) showed that this fraction contained a single band. (h) Sephadex G-200 chromatography (step 8). The procedure was identical to that described in step 7 except that the flow rate was 9 ml/hr and 2-3 ml fractions were collected. The results are shown in Fig. 1. Fractions 51-69 were combined and dialyzed for 12 hr against three changes of 2 1. distilled water each.
Electrophoresis (a) Analytical
polyacrylamide
gel
electrophoresis.
Polyacrylamide gels (7.5~o) were prepared according to the method of BREWER et al. (1974). Electrophoretic separations were carried out on a Shandon Model U 77 electrophoresis cell with a V o k a m constant current/constant voltage power supply at 4°C in a Tris-glycine buffer, pH 8.6. A voltage of 4 mV/gel was applied over the system for about 1 hr. Some of the gels were stained with amidoblack to determine the protein zones while others were used to locate the enzyme zones with lys-p-nitroanilide. (b) SDS-polyacrylamide gel electrophoresis. The gels and protein samples were prepared according to WEBER et al. (1972). A current of 6 mA/gel was maintained until the bromophenol blue zones had migrated to the bottom of the gels. Gels were stained with Kenacid blue R.
Molecular weight determinations The molecular weight of aminopeptidase was determined by making use of gel filtration (ANDREWS. 1970) and SDS-gel electrophoresis (WEBER et al., 1972). For molecular weight estimations a Sephadex G-200 (2.2 cm i.d. x 90 cm) column, equilibrated with 0.15 M KCI solution (ANDREWS, 1965), was calibrated with yeast alcohol dehydrogenase (150,000), bovine serum albumin (67,000), ovalbumin (45,000), ribonuclease (13,500), and cytochrome c (12,500). The molecular mass of the enzyme was also determined by SDS-polyacrylamide gel electrophoresis, as previously described, using SDS molecular weight markers of known molecular mass (56,000-280,000 daltons).
Diffusion coeJ[icient and Stokes radius determinations The data obtained from the elution profile of the Sephadex G-200 column were used as described by ANDREWS(1970) to estimate the diffusion coefficient and Stokes radius of aminopeptidase.
Thin-layer chromatography (TLC) Leu-Gly-Gly, Gly-Gly-Leu and their metabolites were identified by T L C on kieselgel 60F2s 4 plates. The solvent was nbutanol-acetone-Mistilled water (60:15:25, v/v) (SMITH and FEINBURG, 1965) and peptides as well as amino acids were visualized with a ninhydrin spray reagent.
pH optimum and substrate 3pec~ficity The hydrolysis of several substrates at a concentration of 3 m M in 0.02 M Universal buffer (BRITTON and ROBINSON, 1931) at various pH values was determined. In each case the hydrolysis rate at the optimum pH was taken as 100 and the hydrolysis rate at other values expressed as a proportion of this.
Table 1. Specific activity and distribution of aminopeptidase in T. trinervoides workers
Protein (g/l.)
Enzyme activity (units/l. × 10 2)
Specific activity (units/g protein) x 10 -2)
Relative specific activity (~o)
Relative total activity ('?/o)
Foregut Midgut Mixed segment Hindgut 1 Hindgut 2 and 3 Remainder of body
0.618 0.599 2.790 0.358 0.880 1.232
5.741 420.100 126.600 40.020 10.120 14.290
9.29 701.00 45.40 112.00 11.50 11.60
1.0 78.7 5.1 12.6 1.3 1.3
0.9 68.1 20.5 6.5 t.7 2.3
Total
6.477
616.871
890.79
100.0
100.0
Tissue
Termite aminopeptidase
e~ 15--
313
4.0
• Protein
C~-OAminopeptidase
30
I0-20
8
05
V
1.0
13-
40
2O
I~-
~'
60
80
Fraction number
Fig. 1. Elution profile of aminopeptidase on a Sephadex G-200 column. The column (2.2 cm i.d. x 90 cm) was eluted with 0.15 M KCI solution and 2-3 ml fractions were collected. [ ] = Fractions pooled.
Table 2. Purification of aminopeptidase from the termite T. trinervoides
1. 2. 3. 4. 5. 6. 7. 8.
Step procedure
Volume (ml)
Total protein (mg)
Total units
Crude extract 0-30% (NH4)2SO 4 supernatant DEAE-cellulose (1) DEAE--cellulose (2) DEAE --Sephadex Sephadex G-200 CM-Sephadex Sephadex G-200
1250 1400 40 23 10 12 10 5
12,426 9244 2684 836 487 45 38 36
219.00 202.72 111.38 83.12 74.84 67.86 65.94 62.47
tOO I00
Q
--
-.
Specific activity Purification (p/g) (-fold) 17.6 21.9 41.5 99.4 153.7 1508.0 1735.3 1735.3
1.00 1.24 2.36 5.65 8.73 85.78 98.60 98.60
Yield (%) 100.0 92.6 50.9 38.0 34.2 31.0 30.1 28.5
®
G
0
80 80
{
6o
60
40 [3C
20 20
2 Migration distance,
4
6
cm
2 Migrol]on d i s t a n c e ,
Fig. 2. Analytical polyacrylamide gel electrophoresis of purified aminopeptidase. IB 11:3 - E
4
6 cm
Fig. 3. SDS-polyacrylamide gel electrophoresis of purified aminopeptidase.
314
M.C. VAN DER WESTHUIZEN, P. H. HEWITTAND P. J. DU TOIT
25 ~ec~e.,~Ase c \
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tog moleculor mass Fig. 4. Determination of molecular mass of aminopeptidase by Sephadex G-200 gel filtration.
RESULTS
studies (see below) showed that the enzyme was pure and it was used in this form for further studies.
Distribution of aminopeptidase The soluble protein concentrations and the aminopeptidase activities of the various gut regions and the remainder of the insect are given in Table 1.
Purification A peak which hydrolyzed Hammerstein casein slowly but which nevertheless had some activity towards lys-p-nitroanilide was eluted from the DE-52 column before the salt gradient was applied. All 'true' protease activity was thus removed in the initial step. An elution profile of the final purification step is shown in Fig. 1. The combined fractions contained 36 mg of protein and 62.47 units of aminopeptidase representing a final purification of 98.60-fold. Results of a typical purification are given in Table 2. The purification was repeated three times with essentially the same pattern of specific activity increase. The final specific activity was 1735.3 +_ 1.2 units/g protein. The results of the final Sephadex G-200 chromatography (see Fig. 1) and electrophoresis
56~ e
Pentomer
54 r
~eTetramer
Determination of homogeneity Figure 2 is a facsimile of the densitogram of one of the amidoblack stained gels obtained from analytical polyacrylamide gel electrophoresis. The enzyme activity as determined with lys-p-nitroanilide on one of the unstained polyacrylamide gels was confined to a single band, which had exactly the same mobility as the single protein visualized with the amidoblack. The mobility of aminopeptidase from crude midgut extracts had the same mobility as the pure enzyme on acrylamide gel. The densitograph of one of the gels, stained with Kenacid blue R, resulting from SDS-polyacrylamide gel electrophoresis is given in Fig. 3.
Determination of physical properties The molecular weight of aminopeptidase as determined by gel filtration (ANDREWS, 1970) was 88,654 _+ 12.82 and by SDS-polyacrylamide gel (WEBER et al., 1972) was 87,518 4- 10.47. The results are given in Figs. 4 and 5 respectively.
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Rf Fig. 5. Determinationof molecular massof aminopeptidaseby SDS-polyacrylamidegel electrophoresis with molecular mass markers. Protein migration distance Rs = Bromophcnol blue migration distance"
315
Termite aminopeptidase 225
e--e Diffus~n coefficient 0---0 Stokes radius
el
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Ve/Vo Fig. 6. Estimation of diffusion coefficient and Stokes radius by Sephadex G-200 gel filtration. T h e d i f f u s i o n coefficient a n d Stokes radius o f T. were e s t i m a t e d as 5.680 x 10 -7 _+ 0.018 × 10 -7 cm2/sec a n d 37.974 x 10 -8 +__ 0.056 × 10 - s c m respectively. T h e results are given in Fig. 6.
trinervoides a m i n o p e p t i d a s e
Determination of chemical properties (a) pH optimum and substrate specificity. T h e
hydrolysis o f each s u b s t r a t e at different p H values expressed as p e r c e n t a g e o f the m a x i m u m h y d r o l y s i s o f that s u b s t r a t e is given in Table 3. E a c h value r e p r e s e n t s the m e a n o f three replicates. In all cases t h e r e w a s a s h a r p p H o p t i m u m peak, a n d the decline in activity was p a r t i c u l a r l y steep o n the alkaline side o f the o p t i m u m . T h e a b s o l u t e h y d r o l y s i s rate o f each s u b s t r a t e at the p H o p t i m u m
Table 3. Hydrolysis of various substrates at different pH values. Hydrolysis of each substrate is expressed as a percentage of the maximum hydrolysis of that substrate. Standard error is given after each value
pH 2.5 3.0 4.0 5.0 6.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 11.0 12.0
Relative activity Substrate leu-p-nitroanilide lys-p-nitroanilide gly-p-nitroanilide ala-p-napthylamide 0 8 24 40 61 78 88 100 97 91 80 25 12 0
+_ 1.6 + 1.8 +_ 2.1 + 1.4 + 1.0 + 1.7 +_ 1.1 _+ 1.0 _+ 0.8 _+ 1.3 +_ 1.5 _+ 1.1
0 9 25 38 52 67 77 86 96 100 92 55 11 0
+ + + +_ + + + +_ + + _+ +_
1.1 1.7 1.7 1.0 1.4 1.6 1.1 1.0 1.5 1.2 1.4 1.8
0 10 24 38 55 70 85 100 93 31 13 0 0 0
+_ 1.1 + 1.4 + 1.0 + 0.9 +_ 1.6 _ 1.3 + 1.8 _+ 1.1 +_ 3.2 +_ 1.2
0 0 0 0 0 65 80 89 100 70 60 50 0 0
+ 1.6 __ 2.1 + 1.9 + 2.0 +_ 1.8 _+ 1.4 + 1.5
ZTNE 0 0 0 0 14 46 63 77 90 100 87 71 33 0
+ 0.9 _ 1.1 _+ 1.3 + 1.0 _+ 1.5 _ 1.2 +__ 1.0 +_ 1.1 +_ 1.1
BAPA 0 0 14 28 54 81 92 98 100 95 86 75 24 0
+ 2.6 + 1.9 _+ 1.1 + 1.3 + 1.0 + 1.1 +_ 0.8 + 0.8 + 1.0 +_ 1.3 _+ 1.6
Table 4. Substrate specificity of aminopeptidase at optimum pH values
Substrate
Optimum pH
leu-p-nitroanilide 8.0 lysop-nitroanilide 9.0 ZTNE 9.0 Ala-fl-napthylamide 8.5 Gly-p-nitroanilide 8.0 BAPA 8.5 Hydrolysis of casein was negligible at pH 8.5
Degree of hydrolysis and standard error (,umol/ml/min x 10 -z) 18.50 16.25 9.88 8.48 4.92 1.57
+ 0.20 + 0.26 +_ 0.12 + 0.17 +_ 0.09 _+ 0.01
Relative hydrolysis (~) 100 89 53 46 27 8
316
M.C. VAN DER WESTHUIZEN, P. H. HEW1TT AND P. J. DU TO1T
Table 5. Chromatographical separations and identification of products resulting from tripeptide hydrolysis by aminopeptidase Amino acid/peptide
p r o d u c t s of hydrolysis by T L C against a p p r o p r i a t e standards. The results are given in Table 5. The results d e m o n s t r a t e unequivocally t h a t the enzyme is an aminopeptidase. (b) The effects of buJ]er and buJJer concentrations on activity. The hydrolysis of 3 m M lys-p-nitroanilide in each of five buffers over a pH range of 6-10 was determined. The highest activity which was taken as 100 was recorded in p H 9 Barbital buffer. All other activities were expressed as a p r o p o r t i o n of this. The results are given in Table 6. Each value represents the m e a n of three replicates. The lowest activity was recorded in Tris-HC1 buffer which was the only one not containing N a ~ . The effect of increasing ion c o n c e n t r a t i o n of three buffers at pH 8.0 is given in Table 7. Each value represents the m e a n of three replicates. In general enzyme activity increased with increasing ion c o n c e n t r a t i o n reaching a m a x i m u m between 0.2 a n d 0.4 M. Thereafter activity decreased or remained more or less constant. The effects of different c o n c e n t r a t i o n s of metal ions in 0.01 M T r i s - H C l buffer, p H 9.0 on enzyme activity are s h o w n in Fig. 7. Each point represents the m e a n of three replicates. The enzyme was activated by m o n o v a l e n t metals a n d inhibited by divalent metals. The sodium salt of E D T A in 0.01 M Tris-HC1 buffer, p H 9.0, will exchange N a + for divalent ions. This m a y account for the activating effect of E D T A at low concentration. The inhibitory effect at high c o n c e n t r a t i o n s may be due to direct binding with the enzyme. (c) Kinetic constants. The kinetic c o n s t a n t s of the enzyme were determined using lys-p-nitroanilide and
Approximate RI value
Gly Leu Gly-Gly-Leu Leu-Gly-Gly Gly-Gly Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Leu-Gly-Gly Enzyme + Leu-Gly-Gly Enzyme + Leu-Gly-Gly Enzyme
0.21 0.48 0.40 0.35 0.19 0.47 0.21 0.40 0.47 0.19 0.35 0.48 0.00
is presented in Table 4. The rate of hydrolysis of casein, even over a period of 60 min, was negligible. In the light of this observation a n d the results given in Table 4 there can be little d o u b t that the enzyme is an exopeptidase with an alkaline p H o p t i m u m . Long chain synthetic substrates with unsubstituted a - a m i n o groups were most rapidly hydrolyzed. This enzyme was also capable of hydrolyzing the ester g r o u p of Z T N E a l t h o u g h at a comparatively low rate (see Table 4). Carboxy- or amino- terminal preference was determined by incubating the enzyme with 3 m M Leu-Gly-Gly a n d Gly-Gly-Leu separately in 0.02 M T r i s - H C l buffer, p H 8.5, a n d determining the end
Table 6. Relative activity in various buffers at different pH values as a function of optimum activity in Barbital buffer. Standard error is given after each value Barbital 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
77.3 84.0 90.4 96.1 98.7 100.0 98.5
± 0.4 ± 0.2 ± 0.3 ± 0.1 ± 0.5 ± 0.2 +_ 0.1 .
Tris-HCl 78.7 82.7 89.1 91.8 93.9 95.9 94.5 .
± 0.3 ± 0.2 ± 0.5 ± 0.4 +_ 0.1 ± 0.3 ± 0.0 .
Relative activity Phosphate 82.4 84.9 88.6 91.3 94.0 96.6 98.8
Tris-maleate
± 0.4 ± 0.4 ± 0.1 ± 0.3 +_ 0.2 ± 0.5 ± 0.1 -
81.3 84.1 88.6 93.2 96.1 98.7
± ± ± ± ± ± -
Universal
0.3 0.2 0.6 0.4 0.5 0.3
77.6 79.7 84.6 89.9 93.4 96.9 99.2 97.3 89.5
.
± 0.5 +_ 0.4 ± 0.3 + 0.2 ± 0.6 ± 0.3 ± 0.4 + 0.7 i 0.6
Table 7. Relative animopeptidase activity in three buffers at various ion concentrations as a function of optimum activity in phosphate buffer. Standard error is given after each value Buffer concentration (M) 0.005 0.01 0.02 0.05 0.1 0.2 0.3 0.4 0.5 0.6
Tris-HC1 pH 8.0 80.2 82.7 85.2 89.6 92.8 96.1 96.9 97.2 97.2 97.0
± 0.9 +__ 0.4 ± 0.8 ± 0.5 + 0.9 ± 0.6 ± 0.5 ± 0.7 ± 0.4 +__0.5
Relative activity Phosphate pH 8.0 85.0 88.0 92.8 97.2 98.6 100.0 100.0 99.4 98.3 97.3
± ± ± ± ± ± ± ± ± ±
0.4 0.6 0.3 0.9 0.4 0.3 0.5 0.4 0.6 0.3
NaCI dissolved in 5 mM Tris-HCl buffer in pH 8.0 82.5 85.2 91.0 95.0 97.3 98.5 99.4 99.4 99.5 99.6
__+ 0.8 +__ 0.6 ± 0.5 +__0.2 +__ 0.4 +__ 0.5 ± 0.3 ± 0.5 ± 0.6 ± 0.7
Termite aminopeptidase 15(3
AH was estimated as 64.24 _+ 0.013 kJ and AS as 233.81 +_ 0.047 J/°K/mol. In Fig. 12, T In (v/T) was plotted against T yielding a straight line with the equation T I n (v/T) = 28.919 T - 7.882. The slope is given by AS/R. AS was calculated as 240.52 _+ 0.051 J/°K/mol. The values obtained for E,, AH and AS represent the means of three replicates. The Q~o of the enzyme over the temperature range 20°-30°C was determined as 2.59. Figure 13 shows the enzyme activity after a 1 hr exposure to temperature of 3°-80°C. Incubation of the enzyme in the presence of 0.02 M Tris-HC1 buffer, pH 8.0, at 60°C resulted in a 50% loss of catalytic activity at 30°C with 3 m M lys-p-nitroanilide as substrate. Each value represents the mean of three replicates. Incubation under similar conditions at 75°C resulted in total loss of activity.
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DISCUSSION
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M
Fig. 7. Effect o f various metal ions and E D T A , as well as an
increase in ionic strength, on the hydrolysis of 3 mM lysopnitroanilide. Relative activity as a function of the enzymatic hydrolysis in 0.01 M Tris-HCl buffer, pH 9.0. leu-p-nitroanilide (see Fig. 8) as substrates in Tris-HC1 buffer at 30°C. Michaelis constants were estimated as follows. Straight lines were fitted to the data (four replicates/ substrate concentration). The K m values and S.E. at various pH values were as follows: Leu-p-nitroanilide, pH 8.0, K m = 4.023 x l0 -6 _+ 1.1 × 10 -8 M; lys-p-nitroanilide, pH 8.0, K m = 2.048 x 10 -6 + 3 × 10-9 M and lys-p-nitroanilide, pH 9.0, K m = 2.001 × 10 -6 --[- 7 x 10 -Q M. (d) lntiibition studies. A plot of r,:ciprocal of reaction rate (l/v) vs inhibitor concentration at various lys-pnitroanilid,: levels is shown in l~ig. 9. Each poinl reprcs.:nls lh~ mean of three rcplicat,:s. Th,: results arc indicafiv,: ofcomp~fifiv : inhibition. This was confirmed by plotting s/v vs inhibitor concentration and 1/1,vs 1/s both at three subslral,: lcv,:ls. ]h:: K~ as calculav ed from lhesc three plots lay b :tw,L:n 2.1 and 3.0 x 10- 3 M. The K m value of the enzyme determined by Plotting K~ p vs inhibitor concentration was 2.267 x 10 -6 +_ 1.3 x 1 0 8 M a t p H 8 . 5 . (e) Temperature studies. Energy of activation (E~) was calculated as 66.79 kJ from the Arrhenius plot (LAIDLER, 1958) in Fig. 10. A plot of log 100 v against 1000/T yielded a straight line (log 100 v = 12.487 3.488/1000/11 the slope of which is equal to - E,/2.303 R where R is the gas constant. E, was calculated as 66.79 +_ 0.014 kJ. Enthalpy (AH) and entropy (AS) of activation were determined by the methods of CORNISH-BOWDEN (1976) and GUTEREUND (1972). Log (1000 v/11 was plotted against 1000/7', yielding a straight line (Fig. 11) described by the equation log 1000 v/T = 12.5634 - 3.375 1000/T. The slope is equal to log (R/Nh) + AS/2.303 R or - AH/2.303 R where N is the Avogadro's n u m b e r and h is Planck's constant. /
The data in Table 1 show that about 68% of the aminopeptidase activity occurs in the midgut. This is comparable to the results obtained by POTTS and HEWITT (1973) with cellulase, isolated from the same insect and is in agreement with the general pattern of peptide digestion in insects (CHAPMAN, 1971 and HOUSE, 1974). The precise site of action of this enzyme which could be in the cells of the midgut wall, in the space between the midgut wall and the perithrophic membrane or the lumen is unknown. NOIROT and NOIROT-TIMOTHEI~(1969) maintain that digestion in this termite occurs primarily in the hindgut. This statement is not supported by our findings or those of POTTS and HEWITT (1973). Twenty per cent of the aminopeptidase activity was found in the mixed segment. This segment is characterized by the presence of a continuous layer of bacteria, between the peritrophic membrane and the epithelial cells, along its entire length (POTTS and HEWITT, 1973; HONIGBERG, 1970). Since the aminopeptidase activity in the midgut is greater than that in the mixed segment, it seems unlikely that it is produced by the mixed segment bacteria. The purification procedure (see Table 2) yielded a 98.6-fold purification with a final specific activity of 1.75 units/mg of protein. The recovery was 28%. Specific activity values showed that the enzyme was already pure after the seventh (CM-Sephadex) step. This was confirmed by polyacrylamide gel electrophoresis (See Figs. 2 and 3). T. trinervoides aminopeptidase with a molecular mass of 88,000 daltons is considerably smaller than those isolated from Talaromyces duponti, AP 1 (CHAPUIS and ZUBER, 1970), from pig kidney (HIMMELHOCH, 1970 and PFLEIDERER, 1970) from Bacillus stearothermophilus, AP 1 (RONCARI and ZUBER, 1970) and from bovine eye (CARPENTERand VAHL, 1973). The molecular masses of these aminopeptidases are between 280,000 and 400,000 daltons. However, the molecular mass of the T. trinervoides aminopeptidase is in the same order as those of dog brain (MARKSand LAJTHA, 1970), the sisal plant Agave americana (DU TOIT et aL, 1978)
M.C. VAN DERWESTHU1ZEN,P. H. HEWITT AND P. J. DU TOIT
318
24~-- 0 - - • Lys-p-nitroonilide 2o- ~
16--
0 /
>
- 200 (-40)
/ • p H
/ • p H
/ • /
0
8.0
Leu-p-nitroanilide
~
200 (40}
400 (80)
I000 ,
9.0 pH 80
600 (120)
/z/M
q
Fig. 8. Lineweaver-Burk plot ofaminopeptidase with various concentrations oflys- and leu-p-nitroanilide in 0.02 M Tris-HC1 buffer at different pH values. Figures in brackets apply to 1000/s of leu-p-nitroanilide.
T. trinervoides aminopeptidase, in c o m m o n with other aminopeptidases which have been intensively studied (HIMMELHOCH, 1970; MELBYEand CARPENTER, 1971), is active in the pH range from 6 to 11 with maximum activity between 8 and 9. O f the substituted synthetic anilides tested as substrates, the leucine derivative was most rapidly hydrolyzed. This is not in agreement with the results of DE TOIT and SCHABORT (1978a), as reported for a plant aminopeptidase. The N-terminal peptide linkages of Leu-Gly-Gly and Gly-Gly-Leu were rapidly hydrolysed whereas no hydrolysis of C O • H - t e r m i n a l peptide linkages could be demonstrated with T L C techniques. There can thus be little doubt that this enzyme can be classified as an aminopeptidase. The highest activity recorded with the buffers tested was in the concentration range
and the active form of aminopeptidase P (YARON and BERGER, 1970), which have molecular masses in the range 56,000-100,000 daltons. Since the molecular masses as determined by Sephadex G-200 chromatography and SDS electrophoresis are almost identical, it is unlikely that the enzyme consists of more than one protein unit. The diffusion coefficient of T. trinervoides aminopeptidase (5.58 x 10 -7 cm2/sec) does not differ appreciably from those reported for B. stearothermophilus (RONCAR[ and ZUBER, 1970) and A. americana (DU TOrT et al., 1978) with values of 5.0 x 10 -7 and 5.2 x 10 -7 cm2/sec respectively. The Stokes radius of T. trinervoides aminopeptidase (3.8 x 10 _7 cm) is identical to the Stokes radius of A. americana aminopeptidase (DU TOIT et al., 1978).
160
120
"~ 80
4C
I
-I0
0
I0
I
20
i xlO2M
Fig. 9. Product inhibition of aminopeptidase with various concentrations of lysine in 0.02 M Tris-HC1 buffer, pH 8.5. (1 = 3.113 It, 2 = 6.226 # and 3 = 9.340 # lys-p-nitroanilide as substrateL
Termite aminopeptidase
319
~ o
06
I 3 I
I
I 32
i
3.3
3.4
i
35
ICX]OIT
Fig. 10. Arrhenius plot of aminopeptidase with 3 mM lys-p-nitroanilide as substrate in 0.02 M Tris-HC1 buffer, pH 8.0.
•
P
8
(3
I0
I 31
I 32
33
34
:35
10OO/T Fig. 11. Determination of AH and AS with 3 mM lysop-nitroanilide as substrate in 0.02 M Tris-HCl buffer, pH 8.0.
.j.J"
15--
1.3
P ~>
I,.-
I k
jo
Q9
0.7
0.5
I
I
285
293
L ~I Terr~x~r~ure,
I 309
I 317
I 325
°K
Fig. 12. Determination of AS with 3 mM lys-p-nitroanilide as substrate in 0.02 M Tris-HC1 buffer, pH 8.0.
320
M.C. VAN DER WESTHUIZEN, P. H. HEWITT AND P. J. DU TOIT 05
04
03 V
I0
20
30
40
Ternperuture,
50
60
70
°C
Fig. 13. Effect oftemperatureonaminopeptidase. The enzyme was incubated at respective temperatures for 1 hr and thereafter assayed for activity at 30°C.
0.2--0.4 M. The observation that m o n o v a l e n t ions activated a n d divalent ions inhibited has not been reported for other aminopeptidases (CHAPUm and ZUBER, 1970; MARKS a n d LAJTHA, 1970; PFLEIDERER, 1970; YARON a n d BERGER, 1970). The Michaelis c o n s t a n t s for the hydrolysis of leu- and lys-p-nitroanilide by T. trinervoides a m i n o p e p t i d a s e were exceptionally low. The only c o m p a r a b l e Michaelis c o n s t a n t is that for the hydrolysis o f H - P r o - P h e - O H (K m 7.52 × 10 -5) by aminopeptidase P (YARON a n d BERGER, 1970). T. trmervoides lives almost exclusively on a diet of dry grass a n d shrubs (NEE, u n p u b l i s h e d data) b o t h of which have a low protein content. One o f the preferred grasses Themeda triandra contains only 3.5% a m i n o acids (CILLIERS, 1978). The low K m values o f the termite a m i n o p e p t i d a s e m a y be a n a d a p t a t i o n to survival on a low protein content diet. The hydrolysis of dietary protein or bacterial-symbiont protein m a y thus be concerned primarily with the supply of essential a m i n o acids a n d not with the supply of a m i n o acids as foodstuffs. Lysine, one o f the end products o f lys-p-nitroanilide hydrolysis, competes with the substrate for free enzyme. DU TOIT a n d SCHABORT (1978b) made the same observation with A. americana aminopeptidase. The energy a n d enthalpy of activation of termite aminopeptidase is in good agreement with those reported for A. americana aminopeptidase ( o u TOIT a n d SCHABORT, 1978a). The Qlo of termite aminopeptidase (2.59) is for all practical purposes the same as that of A. americana a m i n o p e p t i d a s e (2.58) (DU TOIT and SCHABORT, 1978a).
REFERENCES ANDREWS P. (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606. ANDREWS P. (1970) Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis (Ed. by GUCK D.) Vol. 18, pp. 1-53. John Wiley & Sons, New York. BARMAN T. E. (1969) Enzyme handbook. Vol. 2. Springer, New York. BREWER J. M., PESCE A. J. and ASHWORTH R. B. (1974) Experimental Techniques in Biochemistry. Prentice-Hall, London.
BRITTON H. T. S. and ROBINSON R. A. (1931) Universal buffer solutions and time dissociation constant of veronal. J. chem. Soc. 1456-1462. CARPENTER F. H. and VAHL J. M. (1973) Leucine aminopeptidase (bovine lens). Mechanism of activation by Mg ++ and Mn + + of the zinc metalloenzyme, amino acid composition and sulfhydryl content. J. biol. Chem. 248, 294-304. CHAPMAN R. F. (1971) The Insects." Structure and Function. The English Universities Press, London. CHAPUIS R. and ZUBER H. (1970) Thermophilic aminopeptidases: AP 1 from Talaromyces duponti. In Methods" in Enzymology (Ed. by PERLMANN G, E. and LORAND L.) Vol. 19, pp. 552-555. Academic Press, New York. CILLIERS J. J. LE R. (1978) Seisoenvariasie in die aminosuursamestelling van rumenprotoso~. M.Sc. Thesis. University of the Orange Free State, Bloemfontein. CORNISH-BOWDEN A. (1976) Principles q/" Enzyme Kinetics. Butterworth. London. DI JESO F. (1968) Ammonium sulfate concentration conversion nomograph for OL J. biol. Chem. 243, 2022-2023. DU TOH P. J. and SCHABORT J. C. (1978a) An aminopeptidase from Agave americana, chemical properties of the enzyme. Phytochemistry 17, 371-375. DU TOIT P. J. and SCHABORT J. C. (1978b) An aminopeptidase from Agave americana, thermodynamic studies. Phytochemistry 17, 377-380. DU TOIT P. J., SCHABORTJ. C., KEMPFF P. G. and LAUBSCHER D. S. A. (1978) Aminopeptidase from Agave americana, isolation and physical characterization. Phytochemistry 17, 365-369. GILMOUR G. (1961) The Biochemistry oJlnsects. Academic Press, New York. GOMOR1 G. (1955) Preparation of buffers for use in enzyme studies. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLAN N. O.) Vol. 1, pp. 138-146. Academic Press, New York. GUTFREUND H. (1972) Enzymes." Physical Principles. John Wiley & Sons, New York. HIMMELHOCH S. R. (1970) Leucine aminopeptidase from swine kidneys. In Methods" in Enzymology (Ed. by PERLMANN G. E. and LORAND L.) Vol. 19, pp. 508-513. Academic Press, New York. HONIGBERG B. M. (1970) Protozoa associated with termites and their role in digestion. In Biology of Termites (Ed. by KRISHNA K. and WEISNER F. M.) Vol. 2, pp. 1-36. Academic Press, New York. HOUSE H. L. (1974) Digestion. In The Physiology" o[lnseeta (Ed. by ROCKSTEIN M.) Vol. 5, pp. 63-117. Academic Press, New York. LAIDLER K. J. (1958) The Chemical Kinetics o/ Enzyme Action. Oxford University Press, London.
Termite aminopeptidase LAYNE E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. In Methods in Enzymology (Ed. by COLOWICK S. P. and KAPLAN N. O.) Vol. 3, pp. 447-454. Academic Press, New York. MARKS N. and LAJTHA A. (1970) Brain aminopeptidase hydrolyzing leucylglycylglycine and similar substrates. In Methods in Enzymology (Ed. by PERLMAN G. E. and LORAND L.) Vol. 19, pp. 534-543. Academic Press, New York. MELBYE S. W. and CARPENTER F. H. (1971) Leucine aminopeptidase (bovine lens): Stability and size of subunits. J. biol. Chem. 246, 2459-2463. NOIROT C. H. and NO1ROT-TIMOTHEI~C. (1969) The digestive system. In Biology o f Termites (Ed. by KRISHNA K. and WEXSNERF. M.) Vol. 1, pp. 49-88. Academic Press, New York. PFLEIDERER G. (1970) Particle-bound aminopeptidase from pig kidney. In Methods in Enzymology (Ed. by PERLMANN G. E. and LORAND L.) Vol. 19, pp, 514-521. Academic Press, New York. POTTS R. C. and HEWITT P. H. (1973) The distribution of intestinal bacteria and cellulase activity in the harvester termite Trinervitermes trinervoides (Nasutitermitinae). Insectes soc. 20, 215-220.
321
RONCARI G. and ZUBER H. (1970) Thermophilic aminopeptidases: AP 1 from Bacillus stearothermophilus. In Methods in Enzymology (Ed. by PERLMANNG. E. and LORANO L. ) Vol. 19, pp. 544-552. Academic Press, New York. SMITH I. and FEINBERGJ. G. (1965) Paper and Thin Layer Chromatography and Electrophoresis. Shandon Scientific, London. SPIESJ. R. (1957) Colorimetric procedures for amino acids. In Methods in Enzymology (Ed. by COLOWlCK S. P. and KAPLAN N. O.) Vol. 3, pp. 467-477. Academic Press, New York. WEBER K., PRINGLE J. R. and OSBORN M. (1972) Measurements of molecular weights by electrophoresis on SDS-acrylamide gel. In Methods in Enzymology (Ed. by HIRS C. H. W. and T1MASHEFFS. N.) Vol. 26, pp. 3-28. Academic Press, New York. WIGGLESWORTH V. V. (1965) The Principles of Insect Physiology. Methuen, London. YARON, A. and BERGER A. (1970) Aminopeptidase P. In Methods in Enzymology (Ed. by PERLMANN G. E. and LORAND L.) Vol. 19, pp. 521-534. Academic Press, New York.
0020-1790/81/030311-11502.00/0 © 1981. Pergamon Press Ltd.
AMINOPEPTIDASE FROM THE HARVESTER TERMITE TRINER VITERMES TRINER VOIDES (SJOSTEDT): DISTRIBUTION, PURIFICATION, PHYSICAL AND CHEMICAL PROPERTIES M. C. VAN DER WESTHUIZEN , P. H. HEWITT a n d P. J. DU TOIT* Department of Zoology and Entomology and *Department of Biochemistry, University of the Orange Free State, Bloemfontein, Republic of South Africa (Received 17 April 1980: revised 11 November 1980) Abstract--Approximately 70~o of the digestive aminopeptidase of the harvester termite Trinervitermes trinervoides (Sj6stedt) is found in the midgut. The enzyme was purified using conventional methods. The diffusion coefficient, Stokesradiusandmolecularmassweredeterminedas5.58 x 10-Tcm2/sec, 3.8 x 10 -7 cm and 88,000 daltons respectively. The chemical properties were as follows: pH optimum between 8 and 9, preferred substrate leucine-p-nitroanilide, K m for leu-p-nitroanilide and lys-p-nitroanilide hydrolysis at pH 8.0 were 4.000 x 10-~ M and 2.048 x 10-~ M respectively, Ea, Q l0, AH and AS were 66.79, 2.59 and 64.24 kJ and 232.16 J/°K/mol respectively. Key Word Index: Harvester termite, Trinervitermes trinervoides, aminopeptidase purification, physical and chemical properties
INTRODUCTION THE PRESENCE o f a m i n o p e p t i d a s e has been d e m o n s t r a t e d in several insects (CHAPMAN, 1971; GILMOUR, 1961 a n d WIGGLESWORTH, 1965) but to o u r knowledge n o comprehensive study of any insect a m i n o p e p t i d a s e has yet been undertaken. This l a b o r a t o r y has reported on the properties of cellulase, trehalase a n d aryl-fl-glucosidase from Trinervitermes trinervoides. This p a p e r represents a further c o n t r i b u t i o n to o u r knowledge of digestive enzymes of this termite a n d of insects in general. MATERIALS AND METHODS
The total assay volume was 1.5 ml of 0.02 M Tris-HCl buffer, pH 8.5 containing 3 mM lys-p-nitroanilide and 100/A of enzyme preparation which was added last. Enzyme activity, expressed in/~mole p-nitroanilide released/min/ml, was calculated from the reaction rate during the initial 10 sec. The extinction coefficient of p-nitroanilide used in these calculations was 9.62 x 103 M -1 cm -~ (BARMAN, 1969). The hydrolysis of other amino acid p-nitroanilides was assayed under identical conditions. Ala-fl-napthylamide hydrolysis was determined by the method of LEE et al. (1971) and that of N-carbobenzoxy L-tyrosine-p-nitrophenylester (ZTNE) by the method of BARMAN(1969). Casein hydrolysis was determined by the method of SPins (1957). Distribution of aminopeptidase
Termites Workers and larvae were collected from a mound situated in the vicinity of Bloemfontain. Chemicals Sephadex products were supplied by Pharmacia Chemicals, Sweden. Alcohol dehydrogenase and kiesegel 60F254 plates were obtained from Merck, bovine serum albumin and RNAse from Seravac, SDS molecular weight markers and cytochrome c from BDH and ovalbumien from Miles Biochemicals. All other chemicals, which were supplied by Merck Chemicals, British Drug Houses and ICN Pharmaceuticals, were of analytical grade. Protein determination The spectrophotometric method of Warburg and Christian (LAYNE, 1957), standardised against 2~o (w/v) bovine serum albumin, was used. Enzyme assays Unless otherwise stated all assays were conducted at 30°C. All buffers were prepared according to GOMORI (1955). Aminopeptidase activity was assayed by following the release ofp-nitroanilide, at 405 nm, from lys-p-nitroanilide. 311
The digestive tracts of ten pre-cooled workers (4°C) were removed and cut into the following easily recognisable regions: foregut, midgut, mixed segment, hindgut 1 and hindgut 2 and 3. Mixing of the contents of the various regions was avoided as far as possible. The pooled regions were homogenized with a Potter-Elvejehm homogenizer in 5 ml of 0.03 M Tris-HC1 buffer, pH 8.5. This crude homogenate was centrifuged at 20,000 g for 30 min at 4°C and the supernatant filtered through glass wool to remove fatty material. Purification of aminopeptidase (a) Crude Extract (step I). T. trinervoides workers and larvae (125 g) were homogenized with a Waring blender in 600 ml of ice-cold 0.9~o NaCl solution for 10 min at maximum speed and thereafter for a further l0 min with a high speed top-drive homogenizer. More NaCl (0.9~o) was added to give a total volume of 1.25 I. (10 ml/g wet termites). After stirring at 4°C for 8 hr the slurry was filtered through eight thicknesses of pre-wetted cheese-cloth. The homogenate was centrifuged at 20,000 g for 45 min at 4°C and the supernatant filtered through glass wool to remove the surface lipid layer. After dialysing the supernatant against two changes of 25 I. distilled water for 8 hr, the protein concentration and the enzymatic activity were determined.
312
M . C . VAN DER WESTHUIZEN, P. H. HEWITT AND P. J. DU TOIl
(b) (NH4)2SO 4 fractionation (step 2). The crude supernatant was brought to 30~o saturation with solid (NH,)2SO 4 (DI JESO, 1968). After complete addition of (NH4)2SO 4 stirring was continued for 2 hr. The precipitate was removed by centrifugation at 20,000 g for 45 min at 4°C. The supernatant was filtered through glass wool and dialyzed against five changes of 25 I. distilled water before freeze drying. ( c) DEA E-cellulose chromatography (step 3). The freezedried protein from step 2 was dissolved in 0.02 m Tris-HCl buffer, pH 8.15, and dialyzed for 8 hr against two changes 21. each) of the same buffer. The protein solution was applied to a DEAE-cellulose (DE-52) column (2.2 cm i.d. × 60 cm) equilibrated with the same buffer. Protein was eluted with a linear NaCI gradient (0.0--0.4 M NaCI in 11.) at a flow rate of 21 ml/hr and collected in 10-11 ml fractions. Aminopeptidase containing fractions, (36-56), were combined, dialyzed against three changes (3 1. each) of distilled water for 12 hr and freeze-dried. (d) DEAE-cellulose chromatography (step 4). The protein obtained in step 3 was dialyzed as described in step 3 and then re-chromatographed on a DE-52 column (2.2 cm i.d. x 53 cm) using a 0--0.25 M NaCI gradient in 0.02 M Tris-HCl buffer, pH 8.15. Fractions 45-68 were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. (e) DEAE-Sephadex chromatography (step 5). The step 4 protein was dissolved and dialyzed as described in step 3 and then chromatographed on D E A E - S e p h a d e x A-25 column (1.9 cm i.d. × 28 cm) at a flow rate of 21 ml buffer (0.02 M Tris-HCl, pH 8.15) per hr. Fractions 36---64 were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. (f ) Sephadex G-200 chromatography (step 6). The protein from step 5 was dissolved in 0.15 M KCI solution, dialyzed for 8 hr against 2 L of this solution and fractionated on a 2.2 cm i.d. × 90 cm KCl-equilibrated column at a flow rate of 21 ml/hr. Fractions of 10 ml were collected. Fractions 56-74 were combined, dialyzed against 4 1. distilled water for 8 hr, and then lyophilized. The dried protein was dissolved and dialyzed against 0.016 citrate-phosphate buffer, pH 6.4. (g) CM-Sephadex chromatography (step 7). The dialysate was applied to a 2.2 cm i.d. x 60 cm CM-Sephadex C-25 column equilbrated in the same buffer. The column was developed at a flow rate of 21 ml/hr and 5-6 ml fractions were collected. The aminopeptidase-containi,Lg fractions (8-16) were pooled, dialyzed against 4 1. distilled water for 8 hr and freeze-dried. Gel electrophoresis (pH 8.6) showed that this fraction contained a single band. (h) Sephadex G-200 chromatography (step 8). The procedure was identical to that described in step 7 except that the flow rate was 9 ml/hr and 2-3 ml fractions were collected. The results are shown in Fig. 1. Fractions 51-69 were combined and dialyzed for 12 hr against three changes of 2 1. distilled water each.
Electrophoresis (a) Analytical
polyacrylamide
gel
electrophoresis.
Polyacrylamide gels (7.5~o) were prepared according to the method of BREWER et al. (1974). Electrophoretic separations were carried out on a Shandon Model U 77 electrophoresis cell with a V o k a m constant current/constant voltage power supply at 4°C in a Tris-glycine buffer, pH 8.6. A voltage of 4 mV/gel was applied over the system for about 1 hr. Some of the gels were stained with amidoblack to determine the protein zones while others were used to locate the enzyme zones with lys-p-nitroanilide. (b) SDS-polyacrylamide gel electrophoresis. The gels and protein samples were prepared according to WEBER et al. (1972). A current of 6 mA/gel was maintained until the bromophenol blue zones had migrated to the bottom of the gels. Gels were stained with Kenacid blue R.
Molecular weight determinations The molecular weight of aminopeptidase was determined by making use of gel filtration (ANDREWS. 1970) and SDS-gel electrophoresis (WEBER et al., 1972). For molecular weight estimations a Sephadex G-200 (2.2 cm i.d. x 90 cm) column, equilibrated with 0.15 M KCI solution (ANDREWS, 1965), was calibrated with yeast alcohol dehydrogenase (150,000), bovine serum albumin (67,000), ovalbumin (45,000), ribonuclease (13,500), and cytochrome c (12,500). The molecular mass of the enzyme was also determined by SDS-polyacrylamide gel electrophoresis, as previously described, using SDS molecular weight markers of known molecular mass (56,000-280,000 daltons).
Diffusion coeJ[icient and Stokes radius determinations The data obtained from the elution profile of the Sephadex G-200 column were used as described by ANDREWS(1970) to estimate the diffusion coefficient and Stokes radius of aminopeptidase.
Thin-layer chromatography (TLC) Leu-Gly-Gly, Gly-Gly-Leu and their metabolites were identified by T L C on kieselgel 60F2s 4 plates. The solvent was nbutanol-acetone-Mistilled water (60:15:25, v/v) (SMITH and FEINBURG, 1965) and peptides as well as amino acids were visualized with a ninhydrin spray reagent.
pH optimum and substrate 3pec~ficity The hydrolysis of several substrates at a concentration of 3 m M in 0.02 M Universal buffer (BRITTON and ROBINSON, 1931) at various pH values was determined. In each case the hydrolysis rate at the optimum pH was taken as 100 and the hydrolysis rate at other values expressed as a proportion of this.
Table 1. Specific activity and distribution of aminopeptidase in T. trinervoides workers
Protein (g/l.)
Enzyme activity (units/l. × 10 2)
Specific activity (units/g protein) x 10 -2)
Relative specific activity (~o)
Relative total activity ('?/o)
Foregut Midgut Mixed segment Hindgut 1 Hindgut 2 and 3 Remainder of body
0.618 0.599 2.790 0.358 0.880 1.232
5.741 420.100 126.600 40.020 10.120 14.290
9.29 701.00 45.40 112.00 11.50 11.60
1.0 78.7 5.1 12.6 1.3 1.3
0.9 68.1 20.5 6.5 t.7 2.3
Total
6.477
616.871
890.79
100.0
100.0
Tissue
Termite aminopeptidase
e~ 15--
313
4.0
• Protein
C~-OAminopeptidase
30
I0-20
8
05
V
1.0
13-
40
2O
I~-
~'
60
80
Fraction number
Fig. 1. Elution profile of aminopeptidase on a Sephadex G-200 column. The column (2.2 cm i.d. x 90 cm) was eluted with 0.15 M KCI solution and 2-3 ml fractions were collected. [ ] = Fractions pooled.
Table 2. Purification of aminopeptidase from the termite T. trinervoides
1. 2. 3. 4. 5. 6. 7. 8.
Step procedure
Volume (ml)
Total protein (mg)
Total units
Crude extract 0-30% (NH4)2SO 4 supernatant DEAE-cellulose (1) DEAE--cellulose (2) DEAE --Sephadex Sephadex G-200 CM-Sephadex Sephadex G-200
1250 1400 40 23 10 12 10 5
12,426 9244 2684 836 487 45 38 36
219.00 202.72 111.38 83.12 74.84 67.86 65.94 62.47
tOO I00
Q
--
-.
Specific activity Purification (p/g) (-fold) 17.6 21.9 41.5 99.4 153.7 1508.0 1735.3 1735.3
1.00 1.24 2.36 5.65 8.73 85.78 98.60 98.60
Yield (%) 100.0 92.6 50.9 38.0 34.2 31.0 30.1 28.5
®
G
0
80 80
{
6o
60
40 [3C
20 20
2 Migration distance,
4
6
cm
2 Migrol]on d i s t a n c e ,
Fig. 2. Analytical polyacrylamide gel electrophoresis of purified aminopeptidase. IB 11:3 - E
4
6 cm
Fig. 3. SDS-polyacrylamide gel electrophoresis of purified aminopeptidase.
314
M.C. VAN DER WESTHUIZEN, P. H. HEWITTAND P. J. DU TOIT
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_~e
~ ~ i l 0111
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tog moleculor mass Fig. 4. Determination of molecular mass of aminopeptidase by Sephadex G-200 gel filtration.
RESULTS
studies (see below) showed that the enzyme was pure and it was used in this form for further studies.
Distribution of aminopeptidase The soluble protein concentrations and the aminopeptidase activities of the various gut regions and the remainder of the insect are given in Table 1.
Purification A peak which hydrolyzed Hammerstein casein slowly but which nevertheless had some activity towards lys-p-nitroanilide was eluted from the DE-52 column before the salt gradient was applied. All 'true' protease activity was thus removed in the initial step. An elution profile of the final purification step is shown in Fig. 1. The combined fractions contained 36 mg of protein and 62.47 units of aminopeptidase representing a final purification of 98.60-fold. Results of a typical purification are given in Table 2. The purification was repeated three times with essentially the same pattern of specific activity increase. The final specific activity was 1735.3 +_ 1.2 units/g protein. The results of the final Sephadex G-200 chromatography (see Fig. 1) and electrophoresis
56~ e
Pentomer
54 r
~eTetramer
Determination of homogeneity Figure 2 is a facsimile of the densitogram of one of the amidoblack stained gels obtained from analytical polyacrylamide gel electrophoresis. The enzyme activity as determined with lys-p-nitroanilide on one of the unstained polyacrylamide gels was confined to a single band, which had exactly the same mobility as the single protein visualized with the amidoblack. The mobility of aminopeptidase from crude midgut extracts had the same mobility as the pure enzyme on acrylamide gel. The densitograph of one of the gels, stained with Kenacid blue R, resulting from SDS-polyacrylamide gel electrophoresis is given in Fig. 3.
Determination of physical properties The molecular weight of aminopeptidase as determined by gel filtration (ANDREWS, 1970) was 88,654 _+ 12.82 and by SDS-polyacrylamide gel (WEBER et al., 1972) was 87,518 4- 10.47. The results are given in Figs. 4 and 5 respectively.
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.
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315
Termite aminopeptidase 225
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Ve/Vo Fig. 6. Estimation of diffusion coefficient and Stokes radius by Sephadex G-200 gel filtration. T h e d i f f u s i o n coefficient a n d Stokes radius o f T. were e s t i m a t e d as 5.680 x 10 -7 _+ 0.018 × 10 -7 cm2/sec a n d 37.974 x 10 -8 +__ 0.056 × 10 - s c m respectively. T h e results are given in Fig. 6.
trinervoides a m i n o p e p t i d a s e
Determination of chemical properties (a) pH optimum and substrate specificity. T h e
hydrolysis o f each s u b s t r a t e at different p H values expressed as p e r c e n t a g e o f the m a x i m u m h y d r o l y s i s o f that s u b s t r a t e is given in Table 3. E a c h value r e p r e s e n t s the m e a n o f three replicates. In all cases t h e r e w a s a s h a r p p H o p t i m u m peak, a n d the decline in activity was p a r t i c u l a r l y steep o n the alkaline side o f the o p t i m u m . T h e a b s o l u t e h y d r o l y s i s rate o f each s u b s t r a t e at the p H o p t i m u m
Table 3. Hydrolysis of various substrates at different pH values. Hydrolysis of each substrate is expressed as a percentage of the maximum hydrolysis of that substrate. Standard error is given after each value
pH 2.5 3.0 4.0 5.0 6.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 11.0 12.0
Relative activity Substrate leu-p-nitroanilide lys-p-nitroanilide gly-p-nitroanilide ala-p-napthylamide 0 8 24 40 61 78 88 100 97 91 80 25 12 0
+_ 1.6 + 1.8 +_ 2.1 + 1.4 + 1.0 + 1.7 +_ 1.1 _+ 1.0 _+ 0.8 _+ 1.3 +_ 1.5 _+ 1.1
0 9 25 38 52 67 77 86 96 100 92 55 11 0
+ + + +_ + + + +_ + + _+ +_
1.1 1.7 1.7 1.0 1.4 1.6 1.1 1.0 1.5 1.2 1.4 1.8
0 10 24 38 55 70 85 100 93 31 13 0 0 0
+_ 1.1 + 1.4 + 1.0 + 0.9 +_ 1.6 _ 1.3 + 1.8 _+ 1.1 +_ 3.2 +_ 1.2
0 0 0 0 0 65 80 89 100 70 60 50 0 0
+ 1.6 __ 2.1 + 1.9 + 2.0 +_ 1.8 _+ 1.4 + 1.5
ZTNE 0 0 0 0 14 46 63 77 90 100 87 71 33 0
+ 0.9 _ 1.1 _+ 1.3 + 1.0 _+ 1.5 _ 1.2 +__ 1.0 +_ 1.1 +_ 1.1
BAPA 0 0 14 28 54 81 92 98 100 95 86 75 24 0
+ 2.6 + 1.9 _+ 1.1 + 1.3 + 1.0 + 1.1 +_ 0.8 + 0.8 + 1.0 +_ 1.3 _+ 1.6
Table 4. Substrate specificity of aminopeptidase at optimum pH values
Substrate
Optimum pH
leu-p-nitroanilide 8.0 lysop-nitroanilide 9.0 ZTNE 9.0 Ala-fl-napthylamide 8.5 Gly-p-nitroanilide 8.0 BAPA 8.5 Hydrolysis of casein was negligible at pH 8.5
Degree of hydrolysis and standard error (,umol/ml/min x 10 -z) 18.50 16.25 9.88 8.48 4.92 1.57
+ 0.20 + 0.26 +_ 0.12 + 0.17 +_ 0.09 _+ 0.01
Relative hydrolysis (~) 100 89 53 46 27 8
316
M.C. VAN DER WESTHUIZEN, P. H. HEW1TT AND P. J. DU TO1T
Table 5. Chromatographical separations and identification of products resulting from tripeptide hydrolysis by aminopeptidase Amino acid/peptide
p r o d u c t s of hydrolysis by T L C against a p p r o p r i a t e standards. The results are given in Table 5. The results d e m o n s t r a t e unequivocally t h a t the enzyme is an aminopeptidase. (b) The effects of buJ]er and buJJer concentrations on activity. The hydrolysis of 3 m M lys-p-nitroanilide in each of five buffers over a pH range of 6-10 was determined. The highest activity which was taken as 100 was recorded in p H 9 Barbital buffer. All other activities were expressed as a p r o p o r t i o n of this. The results are given in Table 6. Each value represents the m e a n of three replicates. The lowest activity was recorded in Tris-HC1 buffer which was the only one not containing N a ~ . The effect of increasing ion c o n c e n t r a t i o n of three buffers at pH 8.0 is given in Table 7. Each value represents the m e a n of three replicates. In general enzyme activity increased with increasing ion c o n c e n t r a t i o n reaching a m a x i m u m between 0.2 a n d 0.4 M. Thereafter activity decreased or remained more or less constant. The effects of different c o n c e n t r a t i o n s of metal ions in 0.01 M T r i s - H C l buffer, p H 9.0 on enzyme activity are s h o w n in Fig. 7. Each point represents the m e a n of three replicates. The enzyme was activated by m o n o v a l e n t metals a n d inhibited by divalent metals. The sodium salt of E D T A in 0.01 M Tris-HC1 buffer, p H 9.0, will exchange N a + for divalent ions. This m a y account for the activating effect of E D T A at low concentration. The inhibitory effect at high c o n c e n t r a t i o n s may be due to direct binding with the enzyme. (c) Kinetic constants. The kinetic c o n s t a n t s of the enzyme were determined using lys-p-nitroanilide and
Approximate RI value
Gly Leu Gly-Gly-Leu Leu-Gly-Gly Gly-Gly Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Gly-Gly-Leu Enzyme + Leu-Gly-Gly Enzyme + Leu-Gly-Gly Enzyme + Leu-Gly-Gly Enzyme
0.21 0.48 0.40 0.35 0.19 0.47 0.21 0.40 0.47 0.19 0.35 0.48 0.00
is presented in Table 4. The rate of hydrolysis of casein, even over a period of 60 min, was negligible. In the light of this observation a n d the results given in Table 4 there can be little d o u b t that the enzyme is an exopeptidase with an alkaline p H o p t i m u m . Long chain synthetic substrates with unsubstituted a - a m i n o groups were most rapidly hydrolyzed. This enzyme was also capable of hydrolyzing the ester g r o u p of Z T N E a l t h o u g h at a comparatively low rate (see Table 4). Carboxy- or amino- terminal preference was determined by incubating the enzyme with 3 m M Leu-Gly-Gly a n d Gly-Gly-Leu separately in 0.02 M T r i s - H C l buffer, p H 8.5, a n d determining the end
Table 6. Relative activity in various buffers at different pH values as a function of optimum activity in Barbital buffer. Standard error is given after each value Barbital 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
77.3 84.0 90.4 96.1 98.7 100.0 98.5
± 0.4 ± 0.2 ± 0.3 ± 0.1 ± 0.5 ± 0.2 +_ 0.1 .
Tris-HCl 78.7 82.7 89.1 91.8 93.9 95.9 94.5 .
± 0.3 ± 0.2 ± 0.5 ± 0.4 +_ 0.1 ± 0.3 ± 0.0 .
Relative activity Phosphate 82.4 84.9 88.6 91.3 94.0 96.6 98.8
Tris-maleate
± 0.4 ± 0.4 ± 0.1 ± 0.3 +_ 0.2 ± 0.5 ± 0.1 -
81.3 84.1 88.6 93.2 96.1 98.7
± ± ± ± ± ± -
Universal
0.3 0.2 0.6 0.4 0.5 0.3
77.6 79.7 84.6 89.9 93.4 96.9 99.2 97.3 89.5
.
± 0.5 +_ 0.4 ± 0.3 + 0.2 ± 0.6 ± 0.3 ± 0.4 + 0.7 i 0.6
Table 7. Relative animopeptidase activity in three buffers at various ion concentrations as a function of optimum activity in phosphate buffer. Standard error is given after each value Buffer concentration (M) 0.005 0.01 0.02 0.05 0.1 0.2 0.3 0.4 0.5 0.6
Tris-HC1 pH 8.0 80.2 82.7 85.2 89.6 92.8 96.1 96.9 97.2 97.2 97.0
± 0.9 +__ 0.4 ± 0.8 ± 0.5 + 0.9 ± 0.6 ± 0.5 ± 0.7 ± 0.4 +__0.5
Relative activity Phosphate pH 8.0 85.0 88.0 92.8 97.2 98.6 100.0 100.0 99.4 98.3 97.3
± ± ± ± ± ± ± ± ± ±
0.4 0.6 0.3 0.9 0.4 0.3 0.5 0.4 0.6 0.3
NaCI dissolved in 5 mM Tris-HCl buffer in pH 8.0 82.5 85.2 91.0 95.0 97.3 98.5 99.4 99.4 99.5 99.6
__+ 0.8 +__ 0.6 ± 0.5 +__0.2 +__ 0.4 +__ 0.5 ± 0.3 ± 0.5 ± 0.6 ± 0.7
Termite aminopeptidase 15(3
AH was estimated as 64.24 _+ 0.013 kJ and AS as 233.81 +_ 0.047 J/°K/mol. In Fig. 12, T In (v/T) was plotted against T yielding a straight line with the equation T I n (v/T) = 28.919 T - 7.882. The slope is given by AS/R. AS was calculated as 240.52 _+ 0.051 J/°K/mol. The values obtained for E,, AH and AS represent the means of three replicates. The Q~o of the enzyme over the temperature range 20°-30°C was determined as 2.59. Figure 13 shows the enzyme activity after a 1 hr exposure to temperature of 3°-80°C. Incubation of the enzyme in the presence of 0.02 M Tris-HC1 buffer, pH 8.0, at 60°C resulted in a 50% loss of catalytic activity at 30°C with 3 m M lys-p-nitroanilide as substrate. Each value represents the mean of three replicates. Incubation under similar conditions at 75°C resulted in total loss of activity.
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Fig. 7. Effect o f various metal ions and E D T A , as well as an
increase in ionic strength, on the hydrolysis of 3 mM lysopnitroanilide. Relative activity as a function of the enzymatic hydrolysis in 0.01 M Tris-HCl buffer, pH 9.0. leu-p-nitroanilide (see Fig. 8) as substrates in Tris-HC1 buffer at 30°C. Michaelis constants were estimated as follows. Straight lines were fitted to the data (four replicates/ substrate concentration). The K m values and S.E. at various pH values were as follows: Leu-p-nitroanilide, pH 8.0, K m = 4.023 x l0 -6 _+ 1.1 × 10 -8 M; lys-p-nitroanilide, pH 8.0, K m = 2.048 x 10 -6 + 3 × 10-9 M and lys-p-nitroanilide, pH 9.0, K m = 2.001 × 10 -6 --[- 7 x 10 -Q M. (d) lntiibition studies. A plot of r,:ciprocal of reaction rate (l/v) vs inhibitor concentration at various lys-pnitroanilid,: levels is shown in l~ig. 9. Each poinl reprcs.:nls lh~ mean of three rcplicat,:s. Th,: results arc indicafiv,: ofcomp~fifiv : inhibition. This was confirmed by plotting s/v vs inhibitor concentration and 1/1,vs 1/s both at three subslral,: lcv,:ls. ]h:: K~ as calculav ed from lhesc three plots lay b :tw,L:n 2.1 and 3.0 x 10- 3 M. The K m value of the enzyme determined by Plotting K~ p vs inhibitor concentration was 2.267 x 10 -6 +_ 1.3 x 1 0 8 M a t p H 8 . 5 . (e) Temperature studies. Energy of activation (E~) was calculated as 66.79 kJ from the Arrhenius plot (LAIDLER, 1958) in Fig. 10. A plot of log 100 v against 1000/T yielded a straight line (log 100 v = 12.487 3.488/1000/11 the slope of which is equal to - E,/2.303 R where R is the gas constant. E, was calculated as 66.79 +_ 0.014 kJ. Enthalpy (AH) and entropy (AS) of activation were determined by the methods of CORNISH-BOWDEN (1976) and GUTEREUND (1972). Log (1000 v/11 was plotted against 1000/7', yielding a straight line (Fig. 11) described by the equation log 1000 v/T = 12.5634 - 3.375 1000/T. The slope is equal to log (R/Nh) + AS/2.303 R or - AH/2.303 R where N is the Avogadro's n u m b e r and h is Planck's constant. /
The data in Table 1 show that about 68% of the aminopeptidase activity occurs in the midgut. This is comparable to the results obtained by POTTS and HEWITT (1973) with cellulase, isolated from the same insect and is in agreement with the general pattern of peptide digestion in insects (CHAPMAN, 1971 and HOUSE, 1974). The precise site of action of this enzyme which could be in the cells of the midgut wall, in the space between the midgut wall and the perithrophic membrane or the lumen is unknown. NOIROT and NOIROT-TIMOTHEI~(1969) maintain that digestion in this termite occurs primarily in the hindgut. This statement is not supported by our findings or those of POTTS and HEWITT (1973). Twenty per cent of the aminopeptidase activity was found in the mixed segment. This segment is characterized by the presence of a continuous layer of bacteria, between the peritrophic membrane and the epithelial cells, along its entire length (POTTS and HEWITT, 1973; HONIGBERG, 1970). Since the aminopeptidase activity in the midgut is greater than that in the mixed segment, it seems unlikely that it is produced by the mixed segment bacteria. The purification procedure (see Table 2) yielded a 98.6-fold purification with a final specific activity of 1.75 units/mg of protein. The recovery was 28%. Specific activity values showed that the enzyme was already pure after the seventh (CM-Sephadex) step. This was confirmed by polyacrylamide gel electrophoresis (See Figs. 2 and 3). T. trinervoides aminopeptidase with a molecular mass of 88,000 daltons is considerably smaller than those isolated from Talaromyces duponti, AP 1 (CHAPUIS and ZUBER, 1970), from pig kidney (HIMMELHOCH, 1970 and PFLEIDERER, 1970) from Bacillus stearothermophilus, AP 1 (RONCARI and ZUBER, 1970) and from bovine eye (CARPENTERand VAHL, 1973). The molecular masses of these aminopeptidases are between 280,000 and 400,000 daltons. However, the molecular mass of the T. trinervoides aminopeptidase is in the same order as those of dog brain (MARKSand LAJTHA, 1970), the sisal plant Agave americana (DU TOIT et aL, 1978)
M.C. VAN DERWESTHU1ZEN,P. H. HEWITT AND P. J. DU TOIT
318
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~
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400 (80)
I000 ,
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600 (120)
/z/M
q
Fig. 8. Lineweaver-Burk plot ofaminopeptidase with various concentrations oflys- and leu-p-nitroanilide in 0.02 M Tris-HC1 buffer at different pH values. Figures in brackets apply to 1000/s of leu-p-nitroanilide.
T. trinervoides aminopeptidase, in c o m m o n with other aminopeptidases which have been intensively studied (HIMMELHOCH, 1970; MELBYEand CARPENTER, 1971), is active in the pH range from 6 to 11 with maximum activity between 8 and 9. O f the substituted synthetic anilides tested as substrates, the leucine derivative was most rapidly hydrolyzed. This is not in agreement with the results of DE TOIT and SCHABORT (1978a), as reported for a plant aminopeptidase. The N-terminal peptide linkages of Leu-Gly-Gly and Gly-Gly-Leu were rapidly hydrolysed whereas no hydrolysis of C O • H - t e r m i n a l peptide linkages could be demonstrated with T L C techniques. There can thus be little doubt that this enzyme can be classified as an aminopeptidase. The highest activity recorded with the buffers tested was in the concentration range
and the active form of aminopeptidase P (YARON and BERGER, 1970), which have molecular masses in the range 56,000-100,000 daltons. Since the molecular masses as determined by Sephadex G-200 chromatography and SDS electrophoresis are almost identical, it is unlikely that the enzyme consists of more than one protein unit. The diffusion coefficient of T. trinervoides aminopeptidase (5.58 x 10 -7 cm2/sec) does not differ appreciably from those reported for B. stearothermophilus (RONCAR[ and ZUBER, 1970) and A. americana (DU TOrT et al., 1978) with values of 5.0 x 10 -7 and 5.2 x 10 -7 cm2/sec respectively. The Stokes radius of T. trinervoides aminopeptidase (3.8 x 10 _7 cm) is identical to the Stokes radius of A. americana aminopeptidase (DU TOIT et al., 1978).
160
120
"~ 80
4C
I
-I0
0
I0
I
20
i xlO2M
Fig. 9. Product inhibition of aminopeptidase with various concentrations of lysine in 0.02 M Tris-HC1 buffer, pH 8.5. (1 = 3.113 It, 2 = 6.226 # and 3 = 9.340 # lys-p-nitroanilide as substrateL
Termite aminopeptidase
319
~ o
06
I 3 I
I
I 32
i
3.3
3.4
i
35
ICX]OIT
Fig. 10. Arrhenius plot of aminopeptidase with 3 mM lys-p-nitroanilide as substrate in 0.02 M Tris-HC1 buffer, pH 8.0.
•
P
8
(3
I0
I 31
I 32
33
34
:35
10OO/T Fig. 11. Determination of AH and AS with 3 mM lysop-nitroanilide as substrate in 0.02 M Tris-HCl buffer, pH 8.0.
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jo
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0.7
0.5
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I
285
293
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I 309
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Fig. 12. Determination of AS with 3 mM lys-p-nitroanilide as substrate in 0.02 M Tris-HC1 buffer, pH 8.0.
320
M.C. VAN DER WESTHUIZEN, P. H. HEWITT AND P. J. DU TOIT 05
04
03 V
I0
20
30
40
Ternperuture,
50
60
70
°C
Fig. 13. Effect oftemperatureonaminopeptidase. The enzyme was incubated at respective temperatures for 1 hr and thereafter assayed for activity at 30°C.
0.2--0.4 M. The observation that m o n o v a l e n t ions activated a n d divalent ions inhibited has not been reported for other aminopeptidases (CHAPUm and ZUBER, 1970; MARKS a n d LAJTHA, 1970; PFLEIDERER, 1970; YARON a n d BERGER, 1970). The Michaelis c o n s t a n t s for the hydrolysis of leu- and lys-p-nitroanilide by T. trinervoides a m i n o p e p t i d a s e were exceptionally low. The only c o m p a r a b l e Michaelis c o n s t a n t is that for the hydrolysis o f H - P r o - P h e - O H (K m 7.52 × 10 -5) by aminopeptidase P (YARON a n d BERGER, 1970). T. trmervoides lives almost exclusively on a diet of dry grass a n d shrubs (NEE, u n p u b l i s h e d data) b o t h of which have a low protein content. One o f the preferred grasses Themeda triandra contains only 3.5% a m i n o acids (CILLIERS, 1978). The low K m values o f the termite a m i n o p e p t i d a s e m a y be a n a d a p t a t i o n to survival on a low protein content diet. The hydrolysis of dietary protein or bacterial-symbiont protein m a y thus be concerned primarily with the supply of essential a m i n o acids a n d not with the supply of a m i n o acids as foodstuffs. Lysine, one o f the end products o f lys-p-nitroanilide hydrolysis, competes with the substrate for free enzyme. DU TOIT a n d SCHABORT (1978b) made the same observation with A. americana aminopeptidase. The energy a n d enthalpy of activation of termite aminopeptidase is in good agreement with those reported for A. americana aminopeptidase ( o u TOIT a n d SCHABORT, 1978a). The Qlo of termite aminopeptidase (2.59) is for all practical purposes the same as that of A. americana a m i n o p e p t i d a s e (2.58) (DU TOIT and SCHABORT, 1978a).
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BRITTON H. T. S. and ROBINSON R. A. (1931) Universal buffer solutions and time dissociation constant of veronal. J. chem. Soc. 1456-1462. CARPENTER F. H. and VAHL J. M. (1973) Leucine aminopeptidase (bovine lens). Mechanism of activation by Mg ++ and Mn + + of the zinc metalloenzyme, amino acid composition and sulfhydryl content. J. biol. Chem. 248, 294-304. CHAPMAN R. F. (1971) The Insects." Structure and Function. The English Universities Press, London. CHAPUIS R. and ZUBER H. (1970) Thermophilic aminopeptidases: AP 1 from Talaromyces duponti. In Methods" in Enzymology (Ed. by PERLMANN G, E. and LORAND L.) Vol. 19, pp. 552-555. Academic Press, New York. CILLIERS J. J. LE R. (1978) Seisoenvariasie in die aminosuursamestelling van rumenprotoso~. M.Sc. Thesis. University of the Orange Free State, Bloemfontein. CORNISH-BOWDEN A. (1976) Principles q/" Enzyme Kinetics. Butterworth. London. DI JESO F. (1968) Ammonium sulfate concentration conversion nomograph for OL J. biol. Chem. 243, 2022-2023. DU TOH P. J. and SCHABORT J. C. (1978a) An aminopeptidase from Agave americana, chemical properties of the enzyme. Phytochemistry 17, 371-375. DU TOIT P. J. and SCHABORT J. C. (1978b) An aminopeptidase from Agave americana, thermodynamic studies. Phytochemistry 17, 377-380. DU TOIT P. J., SCHABORTJ. C., KEMPFF P. G. and LAUBSCHER D. S. A. (1978) Aminopeptidase from Agave americana, isolation and physical characterization. Phytochemistry 17, 365-369. GILMOUR G. (1961) The Biochemistry oJlnsects. Academic Press, New York. GOMOR1 G. (1955) Preparation of buffers for use in enzyme studies. In Methods in Enzymology (Ed. by COLOWICKS. P. and KAPLAN N. O.) Vol. 1, pp. 138-146. Academic Press, New York. GUTFREUND H. (1972) Enzymes." Physical Principles. John Wiley & Sons, New York. HIMMELHOCH S. R. (1970) Leucine aminopeptidase from swine kidneys. In Methods" in Enzymology (Ed. by PERLMANN G. E. and LORAND L.) Vol. 19, pp. 508-513. Academic Press, New York. HONIGBERG B. M. (1970) Protozoa associated with termites and their role in digestion. In Biology of Termites (Ed. by KRISHNA K. and WEISNER F. M.) Vol. 2, pp. 1-36. Academic Press, New York. HOUSE H. L. (1974) Digestion. In The Physiology" o[lnseeta (Ed. by ROCKSTEIN M.) Vol. 5, pp. 63-117. Academic Press, New York. LAIDLER K. J. (1958) The Chemical Kinetics o/ Enzyme Action. Oxford University Press, London.
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