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Trypsin-treated Neurospora tryptophan synthetase
J. Mol. Biol. (1964) 9,83-99
Trypsin-treated Neurospora Tryptophan Synthetase: II. Antigenic Properties MICHAEL
D.
GARRICKt AND SIGMUND
R.
SUSKIND
McOollum-Pratt In.'!titute, The John.'! Hopkin.'! University Baltimore, Maryland, U.S.A. (Received 5 March 1964) The antigenic properties of 'I'Sase] that has been digested with trypsin have been examined using antisera prepared against both native and trypsin-treated TSase (digest). Although both types of antisera inhibit all TSase reactions except the synthesis of indoleglycerolphosphate from indole and glyceraldehydephosphate (reaction 3R), the sera may be distinguished by the pattern of inhibition. The equilibrium constant for both the enzyme/anti-enzyme reaction and the enzyme/anti-digest reaction is about 10 - 9 M. Antigenic assays which rely on specific inhibition of TSase suggest that all the antigenic sites of the native enzyme survive tryptic treatment, but with altered combining properties. Although these alterations are detectable with either antiserum, there is a marked dependence upon time for the heterologous . digest/anti-enzyme reaction not found for the homologous digest/anti.digest reaction. Determinations of competition between trypsin-treated and native TSase for antisera indicate that trypsin-treated TSase combines more effectively with anti-digest than with anti-enzyme. Nevertheless, a sufficient excess of digest can also displace native enzyme from an enzyme/anti-enzyme complex. Quantitative precipitin analyses for the digest/anti-enzyme system show that precipitation is not complete. In Ouchterlony agar double-diffusion analyses, trypsin.treated 'I'Sase does not form a precipitin band with either anti-TSase or anti-digest. Nevertheless, digest will block the precipitation of enzyme by antienzyme, provided that the digest and anti-enzyme are initially mixed. The precipitin bands for enzyme/anti-enzyme and enzyme/anti-digest are confluent. These results also are compatible with the conclusion that most of the antigenic sites of the tryptophan synthetase molecule survive tryptic treatment, but with altered combining properties. It is suggested that tryptic treatment of the native enzyme modifies its configuration, and that this modification is reflected in changes in both catalytic properties and the rate of formation of antigen/ antibody aggregates.
1. Introduction Antigenic and enzymic studies of tryptic digests of native highly purified Neurospora crMsa TSase were undertaken as part of a program to relate the structure and function of this protein. Observations have already been presented (Garrick & Suskind, 1964) illustrating the changes in enzymic properties, and showing that the surviving activi-
t Present address: The Moore Clinic, The Jolms Hopkins Hospital, Baltimore, Maryland, U.S.A. t Abbreviations used: TSase, tryptophan synthetase; CRM, cross-reacting material; TSu, tryptophan synthetase unit, defined as one reaction 2 unit (Garrick & Suskind, 1964). 83
84
M. D. GARRICK AND S. R. SUSKIND
ties are properties of an altered enzyme of about the same size as native TSase, but with different electrophoretic mobility. In this paper the effect of trypsin treatment on the antigenic properties of native TSase has been examined with several technics employing homologous and heterologous antisera. The technics include (1) quantitative precipitin analysis; (2) agar gel diffusion; (3) inhibition of the TSase enzyme reactions (Table 1) by antisera; (4) determinations of the ability of trypsin-treated TSase to protect native TSase from specific inhibition by antisera under a variety of conditions. All methods indicate that most TSase antigenic sites survive tryptic digestion, but with altered antibodycombining properties. TA.BLE
1
Reactions catalyzed by wild type N. crassa TSase
+ L-serine ~ L.tryptophan + n-glyceraldohyde-Svphosphate + H 20 (2) Indole + L-serine ~ r.-tryptophan + H 2 0 (3F) Indcle-Scglyoerolphosphate -+ indole + n-glyeeraldehydephosphete (1) Indole-Svglyeerolphosphate
(3R) Indole
+ D-glyceraldehydephosphate -+ indole-3-glycerolphosphate + unidentified
indole derivative
The results for inactivation of TSase enzyme reactions and fractionation of trypsin. treated TSase (Garrick & Suskind, 1964) suggest that digests retaining 1 % of their original reaction 2 activity represent a relatively homogeneous population of molecules. Therefore, trypsin-treated TSase retaining 1% of its original reaction 2 activity was selected as antigen for most of the experiments which are reported here.
2. Methods and Materials (a) Preparation and assay of native and trypsin-treated 'I'Sase
The following procedures are in the preceding paper (Garrick & Suskind, 1964): (a) growth, preparation and fractionation of mycelial extracts of N. crassa; (b) TSase enzyme assays; and (c) reagents and conditions for tryptic treatment of TSase. (b) Antisera
Rabbits were immunized with native or trypsin.treated TSase (R p H fractions) by Freund's adjuvant technic (Cohn, 1952). Three weeks following the initial injection, a single booster dose was administered, and 7 to 11 days later the rabbits were bled and the serum collected. Non-immune serum was obtained prior to immunization. The immunizing doses represented 0'7 to 1·3 mg of pure TSase protein (Garrick, 1963). The sera were heated to 56°C for 30 min prior to use. The heated sera were centrifuged at 12,000 g for 15 min at 4°C, and the clear supernatant solution was carefully separated from the sediment and surface lipid layers by means of a syringe.
(c) Immunological assay based on the specific inhibition of TSase enzyme activities We have employed four different methods which depend on an assay for TSase enzyme activity. Three of the four methods differ only in the order of additions, so that the rate constants for the various reactions involved are responsible for the observed differences. The four assays may be abbreviated as follows, with the order as written indicating the order of additions (writing E for t est enzyme, Ab for antiserum, S for su bst rat e mixture and Ag for antigen (intact enzyme, tryptic digest or cross-reacting material»: (1) inhibition of enzyme activity = E Ab S or E Ab S; (2) absorption of anti-enzyme
+
+
+
+
TRYPSIN -TREATED TRYPTOPHAN SYNTHETASE. II
85
activity = Ab + Ag + E + S; (3) competition for anti-enzyme activity = E + Ag + Ab + S; and (4) displacement of active enzyme from an enzyme/an t i-enzyme complex = Ab + E + Ag + S. Only the concentration of the component in italics is varied. There was a 5-min incubation at 4°C between each addition, excep t between Ag and S for the displacement assay, where the incubation period was deliberately varied .
(1) Inhibition of enzyme activity Anti-enzyme or anti-digest serum (diluted in a solution of 1 rng /ml. bovine plasma albumin in 0·1 M-potassium phosphate] buffer, pH 6·2 or 7·8 dep ending on the enzyme assay conditions) and a sample of enzyme (diluted in a similar fashion) were mixed at 4°C. A substrate mixture was then added, and the assay tube placed at 37°C and assayed as by Garrick & Suskind (1964). Neutralization values are based on the difference in enzyme activity in the presence of equivalent concentrations of antiserum and non-immune serum. One anti-enzyme unit is defined as the amount of antibody which will inhibit one TSu under st anda rd conditions (Suskind, 1957).
(2) Absorption of anti·enzyme activity The method used is essentially that of Suskind (1957). This method was also employed in determining the recovery of 'I'Sase-related antigen during fractionation procedures or supernatant fluid analyses in quantitative precipitin determinations. A standard antienzyme absorption curve was prepared with the original input trypsin.treated TSase as antigen. The quantity of anti-enzyme absorbed by the antigen in a given assay was determined. The recovery of antigen was then calculated by comparison to the standard curve, assuming that tryptic treatment of one TSu results in the formation of one antigen unit.
(3) Competition for anti·enzyme activity Increasing quantities of antigen were added at 4°C to a series of tubes, each of which contained an identical amount of test enzyme (4,0 to 5·0 TSu in various experiments). Subsequently, to each tube an identical quantity of antibody (2'5 to 4'0 anti-enzyme units in various experiments) was added, followed by the addition of substrate. Residual reaction 2 activity was then determined. The TSu ultimately assayed are a measure of the efficiency of enzymically inactive (or less active) antigen in competition with native TSase for anti-enzyme.
(4) Displacement of active enzyme from an enzyme anti-enzyme complex To a series of tubes at 4°C, each containing about 3 anti-enzyme units, approximately 4·5 TSu of test enzyme were added, followed by a quantity of a tryptic digest of TSase that retained about 0·5 of its original 50 TSu. After selected intervals at 4°C, substrate mixture was added. The tubes were then placed at 37°C, and reaction 2 activity was determined. The increase in TSu detected during the period of incubation at 4°C is a measure of the effectiveness of the digest in displacing active TSase from the initial enzyme/anti-enzyme complex.
(d) Quantitative precipitin tests Precipitin reactions were carried out at 4°C for up to 144 hr. The antigen/antibody precipitates were centrifuged at 4°C, washed twice with 0·1 M-potassium phosphate buffer, pH 7'8, and redissolved in 0·25 N-acetic acid. The protein in the precipitates was deter. mined spectrophotometrically at 277 mp' (Gitlin, 1949). The supernatant solutions were tested for antigen and antibody excess as follows: (1) quantitative determination of super. natant enzyme activities (Garrick & Suskind, 1964); (2) quantitative determination of antigen activity by anti-enzyme absorption, using the curve-fitting procedure described
t The potassium phosphate buffer used was a mixture of mono- and di-baaic phosphates depending on the pH required,
86
M. D. GARRICK AND S. R. SUSKIND
in section (c)2 for digest as antigen; (3) quantitative determination of supernatant solution anti-enzyme activity as in section cl; (4) qualitative testing for supernatant solution antigen and antibody by a capillary precipitin method (Kabat & Mayer, 1961).
(e) Double diffUBion in agar A modification of the Ouchterlony technio was used (Oudin, 1952). The gel medium contained 1'5% purified agar, 0'9% sodium chloride, 0'02% methiolate and 0'002% methyl orange. Special Petri plates and agar cutters were purchased from Consolidated Laboratories, Inc. (Chicago, Ill.),
3. Results (a) Inhibition of TSaae enyzme reactions
The anti- digest sera had normal levels of antd-reaction 2 activity. Anti-digest could be distinguished, however, from anti-TSase by the inhibition of TSase reaction 3F. The inhibition of TSase reactions 1 and 3F has been presented in Fig. 1. The degree of inhibition of reactions 1 and 2 is proportional to the quantity of serum added for all three sera except when AblE approaches 1·00. For almost the entire range 100 (b)
(0)
A
A~
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•
A
A
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(d)
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2
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o
2 AblE
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FIG. 1. Inhibition of TSase reactions 1, 2 and 3F by specific anti.sera. The enzyme assays are described in Garrick & Suskind (1964); the prepartion of anti-sera, in Methods and Materials, part (a), and the assay procedure, in Methods and Materials, part (b)l. % E indicates the percentage of the total input enzyme activity detected in the presence of antibody. No significant inhibition or activation of reactions 1, 2 and 3F was detected with equivalent quantities of non-immune sera. AblE indicates the ratio of antibody to TSase (antienzyme units per TSu based on previous assays of the TSase and the antisera). (a) and (b) depict the results for anti-T'Sase; (c) and (d), for one anti-digeat serum; and (e) and (f), for another anti.digest serum. (a), (c) and (e): (0) reaction 2 assays at pH 7'8; (X) reaction 2 assays at pH 6·2. (b), (d) and (f): (.) reaction 1 assays, (fl.) reaction 3F assays.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
87
AblE = 0 to AblE = 1·00,the same proportionality is found for inhibition of reaction 3F by anti-TSase (Fig. l(b». This result confirms earlier observations cited by Yanofsky (1960) and Garrick & Suskind (1963). In contrast, the anti-digest sera are less effective as inhibitors of reaction 3F (Fig. I(d) and (fl). Specific inhibition of reaction 3R by all antisera is very slight. Therefore, it is not surprising that considerable reaction 3R activity is detected in a TSase/anti-TSase specific precipitate. The lack of proportionality noted for inhibition of reaction 2 when AblE approaches 1·00 (Fig. 1(80), (c) and (e» may be accounted for by the disassociation constant of the enzyme/anti-enzyme complex. Using a modification of the method of Samuels (1963), binding constants of 0·25 to 1·10 TSu/ml. have been calculated for three sera (Fig. 2). Based on a value of 120,000 for the molecular weight of TSase and a value of 9
,
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/
.ff:
2
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5
~1l.LJ L,j 4
3
2
o
2
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FIG. 2. Equilibrium constant for the reaction of 'I'Sase with antibody prepared against native or trypsin-treated TSase. (e) results for anti-T'Sase; (., "-) two different anti-digest sera. In the insert, the results are plotted as TSase activity detected (E) ver8U8 TSase activity added (Ex) in the presence of a constant quantity of antisera. In the large Figure the results have been plotted after the theory of Samuels (1963), and values approximating the predicted linearity are obtained. The slopes of the three curves in the large :figure are - 8/TSu; therefore, K = 1/8 TSu.
M. D. GARRICK AND S. R. SUSKIND
88
2200 TSu/mg for the specific activity of pure enzyme, the equilibrium constant for the inhibition of reaction 2 of native TSase by either anti-T'Sase or anti-digest is about 10- 8 . 4 to 10- 9 ' 0 M. (b) Absorption of anti-enzyme activity by trypsin-treated TSase
The general effect of tryptic treatment on the ability of TSase to absorb antienzyme activity is illustrated in Fig. 3. Increasing quantities of native TSase neutralize proportional quantities of anti-enzyme (even in the presence of trypsin and soybean inhibitor). In direct contrast are the results for anti-enzyme absorption by trypsin-treated TSase, where a lack of proportionality is observed. Three approaches I
I
..., (/)
'c::> l1J
E
~2 c:
l1J
I .;:;
c:
-c
a
I
,
0·01
FIG. 3. A comparison of the reactions of native and trypsin-treated TSase with anti-TSase. Preparations used as antigens were taken at the times indicated by the arrows in Fig. l(a) of Garrick & Suskind (1964). The details of the assay procedure and the preparation of the antiTSase will be found in Methods and Materials, parts (b)2 and (a), respectively. 3·8 anti-enzyme units and 4·9 test TSu were used per tube. (A) TSase control; (.) tryptic digest of TSase (t = 180 min). (Redrawn from Garrick & Suskind, 1963.)
have been used to examine the basis for this non-linearity (Figs 4 and 5, Table 2). The results obtained are consistent with the hypothesis that a single new blocking antigen is formed from TSase as a result of tryptic treatment. Furthermore, the antidigest sera may be distinguished from each other and anti-TSase by their rates and extents of reaction with trypsin-treated TSase. (1) Oomparison of a reconstituted mixture of native and trypsin-treated 'I'Sase with partially inactivated trypsin-treated TSase
Trypsin-treated TSase which retains 1% of its original reaction 2 activity fractionates as though all surviving enzymic activities (Garrick & Suskind, 1964) and the
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
89
Il-ntigenic activity are associated with a single protein. In addition, the kinetics of inactivation indicate that the loss of reaction 2 activity very likely results from the hydrolysis of a single peptide bond. Consequently, it is probable that trypsin-treated TSase retaining 51 % of its original reaction 2 activity would contain a digested protein antigenically equivalent to a 50/50 mixture of native TSase and a digest which retains 1% of its original reaction 2 activity. As seen in Fig. 4, this is the case and the results are compatible with the suggestion that the reconstituted mixture and the partially inactivated TSase contain very similar antigens.
• ~o
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c
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MI. of Ag Fro. 4. Comparison of the anti-enzyme absorption properties of partially inactivated TSase and of a mixture of native TSas6 and trypsin-treated TSase. The antigen assay procedure in Methods and Materials, part (b)2, was used. (.) native 'I'Sase control with trypsin and soybean inhibitor present; (0) control tryptic digest of 'I'Sase retaining 1 % of its original reaction 2 enzymic activity; (.) 50/50 mixture of the native TSase and the digest; (0) trypsin.treated TSase retaining 51% of' the original reaction 2 enzymic action.
(2) Kinetics of tryptic alteration of the antigenic properties of 'I'Saee
The results in Fig. 4 suggest that hydrolysis of the peptide bond which inactivates TSase reaction 2 enzymic activity is responsible for the formation of a single antigen with altered antibody-combining properties. In Table 2 the rate of formation of the altered antigen is compared to the rate of tryptic inactivation of reaction 2. The criterion arbitrarily chosen as a measure of the antigenic alteration was the quantity of antigen required to absorb all the anti-enzyme (3,0 units) present in the assay tube. If twice as much digest (relative to native TSase) is required, one may say that it retains 50% of the antigenic activity; if ten times as much, 10%. Since the quantity of trypsin-treated TSase required to absorb all the anti-enzyme is a function of the
90
M. D. GARRICK AND S. R. SUSKIND
quantity of anti-enzyme used, the results can be considered to show that the kinetics for tryptic alteration of TSase antigenic activity are also exponential. TABLE
2
A comparison of the rates of tryptic inactivation of TSaBe reaction 2 enzymic activity and the formation of TSaBe-related antigen Relativet reaction 2 activity
Relativet antigen activity
Square root oft relative reaction 2 activity
1·00 0·37 0·25 0·12 0·083 0·032 0·012 0·010
1·00 0·72 0·48 0·43 0·30 0·19 0·11 0·057
1·00 0·61 0·50 0·35 0·29 0·18 0·11 0·10
The results of several experiments are summarized. In each experiment a TSase R 2 5 _ 31 fraction was incubated with trypsin, tryptic action was halted with soybean inhibitor at a selected time (Garrick & Suskind, 1964), and the reaction 2 activity and antigenic activity were determined. t Relative reaction 2 activity was calculated as the ratio of surviving TSu to input TSu. The square root was selected arbitrarily to emphasize the relationship between the relative reaction 2 activity and the antigenic activity. t Relative antigen activity is the ratio of the quantity of native TSase required to absorb fully the anti-TSase to the quantity of trypsin-treated TSase required to absorb fully the anti. TSase. These quantities were determined from plots resembling Figs 3 and 4.
(3) TirM-dependence of the absorption reaction
In Fig. 5 the rates of reaction of the antisera with native and trypsin-treated TSase are compared employing the absorption assay. There appears to be no increase over a period of 72 hours in the extent of reaction of native TSase with the three sera. In contrast, as reported earlier (Garrick & Suskind, 1963), the digest anti-TSase reaction is time-dependent, reaching its maximum in about 72 hours. It should be noted that the three sera employed could also be distinguished from one another by the rate and extent of reaction with digest. (c) Oompetition between native and trypsin-treated TSaBe for anti-enzyme activity
The CRM's from certain TSase mutants (Suskind, Wickham & Carsiotis, 1963), as well as trypsin-treated TSase (Fig. 3), exhibit non-linear anti-enzyme absorption plots. This lack of proportionality makes quantitative treatment of such results difficult. Another type of antigen assay based on competition between native and altered TSase for anti-enzyme was developed in the hope of finding a theoretical treatment of the results which would permit linear plots and more accurate evaluation of the results. For the assay (see Methods and Materials), one unit of competing antigenic activity is defined as the amount of antigen which competes equally with one TSu of native enzyme for anti-ensyme activity. Then the units of competing antigen per ml. in a given solution may be calculated as follows.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE . II
91
3
L __ll~,...J._~====::r:==~~=t!t===d*~=l/~!-.... II>
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I
....c:
-c
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(c)
2
0·04 MI. of Ag FIG. 6. The time-dependence of the antigen/o.ntibody reaction of native and, trypsin-treated TS!IIle with homologous and heterologous antisera. See Methods and Materials, part (b)2, for experimental details. The order of additions was Ag + Ab + E + S. For the open symbols, the reactants were added about 6 min apart, whereas for the solid symbols there was a 72-hr interval between the addition of Ab and E. (e. 0) results with a native TSase fraction as antigen; ( .... .6.) results with trypsin·treated TSase retaining 1·1 % of its original reaction 2 enzymic activity. (a) depicts the results for anti.TSase; (b), for one anti-digest serum and (c), for another antidigest serum.
Let F
= units of competing antigen per ml, in the antigen sample.
G = TSu/ml. present in the antigen sample, Y = ml. of antigen sample, E T = total concentration of test enzyme (TSu). E = concentration of enzyme detected (TSu), Eo = concentration of enzyme detected with no competing antigen present (TSu).
92
M. D. GARRICK AND S. R. SUSKIND
Then the enzyme detected, E, is (active enzyme) X (fraction of total antigen which is uncombined with anti-enzyme); or,
E
=
(ET
+ GY)(Eo + FY)/(ET + FY).
(1)
This equation is not readily converted into a linear form, but if the antigen has no detectable reaction 2 activity, G = 0, and it becomes: (2)
Rearranging: (3)
Hence, a plot of (E - Eo)/Y verSU8 E - Eo should be linear, with F calculable from either the slope or the intercept. Such plots are linear (Garrick & Suskind, unpublished results) with either td2 CRM, which gives a linear anti-enzyme absorption plot, or td7 CRM, which gives a non-linear anti-enzyme absorption plot. For tryptic digests of TSase, the derivation can be revised as follows. Let J = units of competing antigen per original TSu in the digest sample, g = TSu detected per original TSu in the digest sample, i.e, fractional survival of reaction 2 activity, y = original TSu of digest prior to tryptic treatment. Prior to digestion, one antigen unit equals one TSu, by definition; therefore, the fractional survival of antigen activity, and
E = (E T
+ gy)(Eo + Jy)/(E T + Jy).
J is (I')
It should be possible to neglect g in tryptic digests, where g is small, providing J is of larger magnitude, so, (2')
and rearranging as before (3')
A plot of (E - Eo)/y versus E - Eo for trypsin-treated TSase is given in Fig. 6. The results show that J = 0·53 and g = 0'014, justifying the approximation made in going from equation (I') to (2'). Using this method, determinations with anti-TSase sera suggest that 20 to 50% of the antigenic activity has been retained in different digest preparations with g = 0·005 to 0·02. Similar determinations with one anti-digest serum give values of 40 to 60% for antigenic survival; and for another, 80 to 120%. (d) Displacement oj active TSase from a TSase/anti-TSase complex
It has been observed that trypsin-treated TSase competes effectively with native TSase for anti-enzyme activity (part C) and that there is an experimentally detectable equilibrium for the TSase/anti-TSase reaction (part (a)). Therefore, trypsin-treated TSase should displace native TSase from an antigen/antibody complex and release reaction 2 activity. Table 3 shows that a sufficient excess of trypsin-treated TSase frees at least 30% of the native TSase that had been previously inhibited by antiTSase.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
93
4
o
3
1!------~~---____;~----~\_r_----~~ 0·05 0·20 o
0·5 (b)
0·4
fl~ 0·2
0·\
o
2
4
E-E o (TSu)
FIG. 6. Competition between native and trypsin-treated 'I'Sase for anti-TSase. For experimental details see Methods and Materials, part (b)3. In (a) the experimental points are plotted directly as 'I'Sase reaction 2 enzymic activity detected ver81Ul ml, of the antigen, trypsin-treated TSase retaining 1·4% of its original reaction 2 activity (250 'I'Sujml.), The smooth curve was calculated assuming that the trypsin-treated TSase retained 50% of its original capacity to compete for anti-enzyme, In (b) the results have been recalculated according to equation (3') in the text. The limits have been placed assuming the error of the reaction 2 assay is ± 0·2 TSu. Both the intercept and the slope indicate that slightly more than 50% of the competing antigenic activity survived digestion. The intercept value is 0'36, therefore f = 0·52; the slope is - 0·116 and therefore J= 0-53.
94
M. D. GARRICK AND S. R. SUSKIND TABLE
3
Displacement by trypsin-treated TSase of active TSase from a TSase/anti-TSase complex Addition
Controls E E+Ab (Ag +Ab) + E iE +Ag Experimental (E +Ab) +Ag Controls E E +Ab (Ag +Ab) +E Experimental (E +Ab) +Ag
0
0·2
4·42 1·57 4·30 2·95
4·32 1·58 4·29 2·97
1·0
2·31
Time in hours 4·0 6·5
28
54
TSu detected 4·34 4'39 1·08 1-13 4·26 4·24 2·97 2·68
4·03 0·97 3·71 1·32
2·71
2·76
3·08
127
73·5
4·50 1·41 4·50
4·25 1·44 4·58
4'36
4·31
4·31
4·25
4·17
3·97
1·91
2·50
2·47
2·81
2·92
For experimental details see Methods and Materials part (b}4. The results of two separate experiments are summarized. E = native TSase, Ab = anti-TSase, Ag = trypsin-treated TSase (in each experiment, original reaction 2 activity before digestion was 50 TSu). The zero-time values are based on prior assay of the antibody, test enzyme and digest. In each of the controls, the TSu detected remains the same or slowly declines during storage at 4°C. In contrast, the reaction 2 activity in the experimental tubes increases considerably before declining, indicating that active TSase has been displaced from an antigen/antibody complex. TABLE
4
Amount of precipitin for native TSase/anti-TSase and trypsin.treated TSase/anti.TSase incubated at 4°0 for 144 hr Protein Native Anti· precipiTSase enzyme tated (TSu) units (p.g) 10 20 30 40 50 60 70 80 90 100 110 120 130 150 200 50 150
67 67 67 67 67 67 67 67 67 67 67 67 67 67 67
49 90 136 158 192 198 178 112 111 85 82 77 46 50 41 o (non- 5 Oim12 mune)
Supernatant antigen Units for TSase reaction 3F t 3Rt 2(TSu) 1 0 0 0 0 0 0 0·04 0·09 0·18 0·54 0·60 0·65 0·80 HO 1·70 0·75 2·30
0 0 0 0 0 0 4 7 15 25 40 46 59 63 90 40 95
0 0 0 0 0 0 1 5 9 27 33 38 56 70 130 51 150
0 0 0 0 0 0 1 2 6 20 19 20 22 27 38 12 46
Units Capilof lary antigen test 0 0 0 0 0 0 5 10 15 36 38 40 54 83 120 60 140
0 0 0 0 0 0 0 0 0
± + + + ++ ++ + ++
Supernatant antibody Anti- Capilenzyme lary test units 44 19 9 3 0 0 0 0 0 0 0
+ + 0 0 0 0 0 0 0 0 0 0 0 0 0
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II TABLE
Trypsin Protein treated Antd- precipienzyme TSase tated (original units (,.g) TSu) 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67
37 74 107 119 96 127 48 74 66 71 62 60 61 64 57 o (non- 7 O(im- 13 mune)
10 20 30 40 50 60 70 80 90 100 110 120 130 150 200 50 150
4 (cont.)
Supernatant antigen Units for TSase reaction 3Ft 3Rt 0 0 0 0 0 0 0·04 0·05 0·06 0·10 0·11 0·15 0·19 0·26 0·34 0·15 0·45
1 2 3 4 5 5 7 12 17 19 20 28 36 38 49 18 53
95
Units of antigen 0 0 0 0 0 0 5 7 14 29 33 48 54 82 120 64 160
Oapillary test 0 0 0 0 0 0 0 0 0
+ + + + ++ ++ + ++
Supernatant antibody AntiCapilenzyme lary units test 40 21 9 8 5 5 4 4 2 4 2 3 3 4 2
+ +0 0 0 0 0 0 0 0 0 0 0 0 0
See Methods and Materials, part (d), for experimental details. A TSase R 2 8 _ 31 fraction was used for both the native and trypsin-treated TSase. The latter retained 0·8% of its original activity for reactions 1 and 2, 20% of its original reaction 3F activity and 54% of its original reaction 3R activity. All values are the mean of at least two determinations. t To increase the sensitivity of the reaction 3F assay, the reaction mixture was incubated for 5 hr at 37°C instead of the usual 1 hr (Garrick & Suskind, 1964). t Assayed at pH 6·2.
(e)
Quantitative precipitin analysisof native and trypsin-treated TSase
Quantitative precipitin observations for the homologous (native TSase) and heterologous (trypsin-treated TSase) reactions with anti-TSaso are summarized in Table 4 and Fig. 7. Although there is considerably less precipitate in the antibody excess and equivalence zones of the heterologous system, the equivalence zone for each reaction is 60 to 70 original TSu on the basis of maximum precipitation and analyses of supernatant solution. Nevertheless, it should be noted that with trypsin-treated TSase as antigen, reaction 3R activity is detected even in supernatant solutions containing excess antibody and small quantities of anti-enzyme activity are observed in supernatant solutions containing excess antigen. In quantitative precipitin deter. minations with the trypsin-treated TSase/anti-TSase'reaction, considerable variation in the maximum quantity of protein precipitated has been observed, depending on the serum used and the reaction time at 4°0. These observations are compatible with the survival of most TSase antigenic sites, but suggest that trypsin has altered the combining properties with antibody. A structural modification of the enzyme, resulting in reduced precipitability of the antigen.antibody complexes, would account for the reaction 3R activity which remains in the supernatant solutions of the antibodyexcess region as well as the anti- TSase found in the region of antigen-excess.
96
M. D. GARRICK AND S. R. SUSKIND 200
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'u <1l
o,
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o
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o
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100
o
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FIG. 7. Quantitative precipitin results for the native TSaBe/anti-TSase and trypsin-treated TSase/anti.TSaBe system. See Table 4 for the results in tabular form. (0) native TSase/anti-TSaBe system; (.) brypsln-teeated TSase/anti-TSaBe system. (0) theoretical plot for the trypsin-treated 'I'Saeejanti-T'Saee system, The theoretical points were calculated from the data in Table 4 as (quantity of specific precipitate for the native TSase/anti-TSaBe system) X (% of reaction 3R activity precipitated for the trypain-treeted TSase!anti-TSaBe syBtem)/(% of reaction 3R activity precipitated for the native TSasejanti-TSaBe system),
Maximum precipitation of one TSasp/anti.digest system is about 60% of the value obtained with the 'I'Sasefanti-T'Saee system when the two are compared on the basis of equivalent anti-enzyme units. In spite of the fact that the anti-digest system is slightly contaminated by additional antigen-antibody reactions, the diminished quantity of precipitate indicates that the two sera do not possess identical specificities for TSase. (f) Agar double-diffusion analY8is
Analyses by double diffusion in agar are depicted in Plate 1. Plate I(A) demonstrates loss of precipitability as a result of tryptic treatment of TSase. Nevertheless, the antigenic activity of trypsin-treated TSase can still be detected with this technic (Plate I(B», since mixing the antiserum and the digest in a single reservoir prevents the subsequent formation of a 'I'Sasejanti-TSase precipitin band. Plate 1(0) illustrates the confluent reaction of anti-TSase and anti-digest with native TSase.
4. Discussion (a) What fraction of the antigenic sites of TSase survive tryptic treatment?
In considering the above question, it is important to keep in mind a key difference between the antigenic determinations based on precipitin formation and the assays based on inhibition of TSase reaction 2. Specifically, the results which rely on inhibition of enzymic activity (in parts (a) to (d) depend primarily on the initial combination of TSase with antibody so as to inhibit TSase enzymic activity, whereas those involving precipitin determinations (in parts (e) and (f) depend on both the initial rapid formation of antigen/antibody complexes and the subsequent slower formation of larger insoluble antigen/antibody aggregates. The fraction of the precipitating antibody which specifically inhibits reaction 2 may be calculated from the quantitative
PLATE 1. Ouchterlony agar diffusion analysis of TSase, tryptic digests of TSase, anti-TSase and anti-digest in heterologous and homologous combinations. See Methods and Materials, part (d), for details of the procedure. (A) The center reservoir contained 240 anti-enzyme unitsjrnl. Reservoir 1 contained 250 'I'Sujml, of an R pH fraction (Garrick & Suskind, 1964); reservoirs 2 and 6, 250 'I'Sujrnl, of the same fraction in the presence of trypsin and soybean inhibitor; reservoir 3, the same concentration of TSase after tryptic inactivation to 23% of its original reaction 2 activity; reservoir 4, the same concentration of TSase after tryptic inactivation to 0·9% of its original reaction 2 activity; and reservoir 5, the same ooneentrat.ion of TSase after tryptic inactivation to 0·4% of its original reaction 2 activity. Similar results were observed for the major precipitin band with all other anti-enzyme and anti-digest sera. Additional bands were observed for all reservoirs with antidigest sera, however. (B) The center reservoir contained 120 anti-enzyme unitsjrnl. Reservoirs 1 and 4 contained 110 TSu/ml. of an R p H fraction in the presence of trypsin and soybean inhibitor; reservoirs 2 and 5, the same TSase after tryptic inactivation to 1 % of its original reaction 2 activity at a concentration that represents 280 original input TSu/mI., and reservoirs 3 and 6, the same digest as in reservoirs 2 and 5 (final concentration equivalent to 140 original input TSu/mI.), mixed with anti-enzyme (final concentration 120 anti-enzyme unit.s/rnl.). Similar results were observed with the other sera. (0) The center reservoir contained 120 'I'Sujrnl, of an R p H fraction. Reservoir 2 contained 270 anti-enzyme unitsjml. of one anti-digest; reservoir 4, 350 anti-enzyme unitejrnl, of another anti-digest; and reservoir 5, 500 anti-enzyme unitsjml. of anti-TSase. The other-three reservoirs contained other sera that do not concern us here.
[facing p. 96
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
97
precipitin observations, assuming that the neutralizing antibody possesses two identical combining sites and that each molecule of enzyme possesses one active site totally inhibitable after combination with antibody. Based on values of 160,000 for the molecular weight of the antibody, of 120,000 for the molecular weight of TSase, and of 2200 TSu/mg for the specific activity of pure TSa se, the 30 to 33 fLg of TSase in the equivalence precipitate combines with 20 to 22 fLg of neutralizing ant ibody and 160 to 170 p.g of precipitating antibody. Hence, only about 13% of the precipitating anti. TSase specifically inhibits TSase. For different determinations on various sera, values of 12 to 25% have been obtained. For comparison, Samuels (1963) finds that 15% of rabbit anti-creatine kinase is neutralizing antibody. Thus, when considering the a.ssays in the Results, parts (b) to (d), it should be kept in mind that these observations probably depend on the presence of no more than one-quarter of the antibody population. The several types of results presented in this paper cannot be accounted for unless a sizable fraction of the antigenic sites survives tryptic treatment. The simplest way to account for all the immunochemical results is to assume that all the antigenic sites are retained, and that the apparent discrepancies measure alterations in the antibodycombining properties of TSase. (b) What is the size oj the antigen present in trypsin-treated TSase?
The gel fractionations described in Garrick & Suskind (1964) were also monitored for antigenic activity by the absorption assay (Methods and Materials , part (b)2). The profile for antigenic activity was identical to the profiles for the surviving enzymic reactions on Sephadex G-75, G-I00 and G-200. For this reason it is concluded that the molecular weight of the antigenically active residual protein of t rypsin-t reat ed TSase is about the same size as the native enzyme (molecular weight = 120,000 (Carsiotis , Appella & Suskind, manuscript in preparation)). Precipitin results reported earlier (Garrick & Suskind, 1963) suggested that tryptic digests of TSase retain only 50 to 80% of th e initial antigenic activity of the enzyme and that the precipitating antigen in the digest had a maximum molecular weight of about 30,000. As initially emphasized, the survival value and the molecular weight limit were calculated on the assumption that aU of the antigen and antibody were precipitated at equivalence. The additional supernatant solution analyses in Table 4 indicate that this assumption is not justified and that the earlier interpretation of the results is invalid. (c) Is trypsin-treated 'I'Saee a successJul immunizing antigen?
Trypsin-treated TSase elicits the formation of antibody in rabbits. Because the immunizing antigen still retained 1·5 to 1·9% of the original reaction 2 activity (= 30 to 68 TSu), the possibility remains that this residual activity, representing a maximum of 13 to 31 fLg of native TSa se, was the actual immunizing antigen. Two lines of evidence rule out this possibility. First, it has been argued (Garrick & Suskind, 1964) that the residual TSase reaction 2 activity after about 99% inactivation by trypsin is not a property of the intact molecule . Second, several results in this paper indicate that the anti-digest sera are not identicalin specificity to the anti-TSase sera: (1) one anti-digest serum may be readily distinguished from anti·TSase by means of the competition assay (Results, section (cj); (2) the time-dependence observed for the extent of absorption of anti-TSase anti-enzyme activity by digest is markedly reduced (Fig. 5) when compared with the absorption of the anti-enzyme activity of the two 7
98
M. D. GARRICK AND S. R. SUSKIND
anti-digest sera; (3) when compared on the basis of anti-enzyme units, anti-TSase inhibits TSase reaction 3F enzymic activity more effectively than either anti-digest serum (Fig. 1); (4) on the basis of JLg protein precipitated per anti-enzyme unit, one anti-digest serum contains less precipitating antibody than the anti-TSase sera. Therefore, trypsin-treated TSase induces the formation of antibodies which are similar, but not identical, to anti-TSase. (d) How does trypsin modify TSase antigenic properties?
It is likely that most of the differences between the reaction of anti-TSase with trypsin-treated TSase and the reaction with native TSase are caused by a somewhat reduced affinity constant and a defect in formation of antigen/antibody aggregates. The trypsin-induced reduction in affinity constant of antigen for antibody is invoked to account for the competition results. Such an alteration combined with a reduced precipitability of antigen/antibody complexes would account for the antibody analyses of supernatant solutions in Table 4. The best evidence for a defect in the aggregation of initial antigen/antibody complexes comes from the precipitin results, where it may be seen that less precipitate is found with the trypsin-treated 'I'Sasejanti-T'Sase system than with the native TSase/anti-TSase system. In addition, the lack of an agar diffusion precipitin band for the trypsin-treated TSase/anti-TSase reaction may also be attributed to such a. defect. It is likely that the time-dependent increase in extent of the digest/anti-TSase reaction recorded in Fig. 5 is also a result of the slow formation of digest/anti-TSase aggregates. Another trypsin-induced alteration in the antigenic properties of TSase is the nonlinearity of anti-enzyme absorption (Fig. 3). This modification is not readily accounted for by either an "alteration in affinity constant or a defect in antigen/antibody aggregation. The non-linearity is not due wholly to a trypsin-induced alteration in the affinity constant for the enzyme/anti-enzyme reaction, since the lack of proportionality is also observed (Fig. 5) for a serum where competition assays detect no change in this constant. In addition, it is unlikely that the lack of proportionality is due wholly to a defect in antigen/antibody aggregation, because a non-linear absorption plot is found when digest has been incubated with anti-TSase for 72 hours (Fig. 5) or longer. This lack of proportionality in the absorption plot for trypsin-treated TSase is of special interest since the mutationally altered TSase of many TSase mutants (e.g. td 7 and td 24 ) also exhibit this property (Suskind et al., 1963). Recent unpublished evidence from this laboratory suggests that N. crassa TSase is composed of several subunits. Perhaps a trypsin-induced alteration in quaternary structure is responsible for the non-linearity of the absorption plot. It seems likely that tryptic treatment of TSase could prove very useful in the study ofimmunological properties ofthe CRM's and CRM-Iess extracts (Suskind et al., 1963). In particular, trypsin-treated TSase may contain materials which would serve as models for the "blocking" activity of certain CRM-less mutants or for the CRM's giving non-linear anti-enzyme absorption plots. (e) The 'I'Sase system for immunochemical studies
(1) It has been suggested that no more than one-quarter of the precipitating antibodies for TSase inhibit reaction 2. This result is very encouraging in the search for TSase-related materials in the CRM-negative TSase mutants, since it demonstrates
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
99
that the absence of CRM may not rule out the presence of up to about 75% of the TSase antigenic sites. (2) Reactions 2, 3F and 3R do not exhibit identical antibody inhibition plots after initial reaction with antisera. Results for reaction 2 may be used for accurate quantitative determination of the enzyme/anti-enzyme reaction, since this activity can be fully inhibited. Because reaction 3R is not completely inhibited, TSase activity in antigen/antibody precipitates may be determined. (3) It is possible to measure the reaction of TSase-related antigen with antibody by assaying for several different enzymic activities, in addition to employing conventional immune-chemical techniques. Therefore, heterologous and homologous combinations of native, trypsin-treated and mutationally altered TSase with respective antisera might also serve usefully in studying antigen/antibody reactions in general, i.e. the nature of the precipitin reaction, both in quantitative precipitin analyses and during diffusion in agar. (4) The equilibrium constant for disassociation of the TSase/anti-TSase complex involved in the specific inhibition of TSase reaction 2 enzymic activity has been determined as 10- 8·4 to 10- 9·0 M. This is one of the few available determinations for a protein/anti-protein reaction. This investigation was performed while one of us (M.D.G.) was a predoctoral fellow of the U.S. Public Health Service. The research was supported by grant number C·03080 of the National Cancer Institute, National Institutes of Health. This paper is contribution number 411 of the McCollum-Pratt Institute. REFERENCES Cohn, M. (1952). Methods Med. Res. 5, 268. Garrick, M. D. (1963). Ph.D. Thesis. The .Iohna Hopkins University, Baltimore, Maryland. Garrick, M. D. & Suskind, S. R. (1963). Ann. N.Y. Acad. Sci. 103, 793. Garrick, M. D. & Suskind, S.,R. (1964). J. Mol. Biol. 9, 70. Gitlin, D. (1949). J. Immunol, 62, 437. Kabat, E. A. & Mayer, M. M. (1961). Experimentalimmunochemistry. Springfield, Illinois: Charles C. Thomas. Oudin, .T. (1952). Methods Med. Res. 5, 858. Samuels, A. (1963). Ann. N.Y. Acad. Sci. 103, 858. Suskind, S. R. (1957). J. Bact. 74, 308. Suskind, S. R., Wickham, M. L. & Carsiobis, M. (1963). Ann. N.Y. Acad. Sci. 103. 1106. Yanofsky, C. (1960). Bact. Rev. 24, 221.
Trypsin-treated Neurospora Tryptophan Synthetase: II. Antigenic Properties MICHAEL
D.
GARRICKt AND SIGMUND
R.
SUSKIND
McOollum-Pratt In.'!titute, The John.'! Hopkin.'! University Baltimore, Maryland, U.S.A. (Received 5 March 1964) The antigenic properties of 'I'Sase] that has been digested with trypsin have been examined using antisera prepared against both native and trypsin-treated TSase (digest). Although both types of antisera inhibit all TSase reactions except the synthesis of indoleglycerolphosphate from indole and glyceraldehydephosphate (reaction 3R), the sera may be distinguished by the pattern of inhibition. The equilibrium constant for both the enzyme/anti-enzyme reaction and the enzyme/anti-digest reaction is about 10 - 9 M. Antigenic assays which rely on specific inhibition of TSase suggest that all the antigenic sites of the native enzyme survive tryptic treatment, but with altered combining properties. Although these alterations are detectable with either antiserum, there is a marked dependence upon time for the heterologous . digest/anti-enzyme reaction not found for the homologous digest/anti.digest reaction. Determinations of competition between trypsin-treated and native TSase for antisera indicate that trypsin-treated TSase combines more effectively with anti-digest than with anti-enzyme. Nevertheless, a sufficient excess of digest can also displace native enzyme from an enzyme/anti-enzyme complex. Quantitative precipitin analyses for the digest/anti-enzyme system show that precipitation is not complete. In Ouchterlony agar double-diffusion analyses, trypsin.treated 'I'Sase does not form a precipitin band with either anti-TSase or anti-digest. Nevertheless, digest will block the precipitation of enzyme by antienzyme, provided that the digest and anti-enzyme are initially mixed. The precipitin bands for enzyme/anti-enzyme and enzyme/anti-digest are confluent. These results also are compatible with the conclusion that most of the antigenic sites of the tryptophan synthetase molecule survive tryptic treatment, but with altered combining properties. It is suggested that tryptic treatment of the native enzyme modifies its configuration, and that this modification is reflected in changes in both catalytic properties and the rate of formation of antigen/ antibody aggregates.
1. Introduction Antigenic and enzymic studies of tryptic digests of native highly purified Neurospora crMsa TSase were undertaken as part of a program to relate the structure and function of this protein. Observations have already been presented (Garrick & Suskind, 1964) illustrating the changes in enzymic properties, and showing that the surviving activi-
t Present address: The Moore Clinic, The Jolms Hopkins Hospital, Baltimore, Maryland, U.S.A. t Abbreviations used: TSase, tryptophan synthetase; CRM, cross-reacting material; TSu, tryptophan synthetase unit, defined as one reaction 2 unit (Garrick & Suskind, 1964). 83
84
M. D. GARRICK AND S. R. SUSKIND
ties are properties of an altered enzyme of about the same size as native TSase, but with different electrophoretic mobility. In this paper the effect of trypsin treatment on the antigenic properties of native TSase has been examined with several technics employing homologous and heterologous antisera. The technics include (1) quantitative precipitin analysis; (2) agar gel diffusion; (3) inhibition of the TSase enzyme reactions (Table 1) by antisera; (4) determinations of the ability of trypsin-treated TSase to protect native TSase from specific inhibition by antisera under a variety of conditions. All methods indicate that most TSase antigenic sites survive tryptic digestion, but with altered antibodycombining properties. TA.BLE
1
Reactions catalyzed by wild type N. crassa TSase
+ L-serine ~ L.tryptophan + n-glyceraldohyde-Svphosphate + H 20 (2) Indole + L-serine ~ r.-tryptophan + H 2 0 (3F) Indcle-Scglyoerolphosphate -+ indole + n-glyeeraldehydephosphete (1) Indole-Svglyeerolphosphate
(3R) Indole
+ D-glyceraldehydephosphate -+ indole-3-glycerolphosphate + unidentified
indole derivative
The results for inactivation of TSase enzyme reactions and fractionation of trypsin. treated TSase (Garrick & Suskind, 1964) suggest that digests retaining 1 % of their original reaction 2 activity represent a relatively homogeneous population of molecules. Therefore, trypsin-treated TSase retaining 1% of its original reaction 2 activity was selected as antigen for most of the experiments which are reported here.
2. Methods and Materials (a) Preparation and assay of native and trypsin-treated 'I'Sase
The following procedures are in the preceding paper (Garrick & Suskind, 1964): (a) growth, preparation and fractionation of mycelial extracts of N. crassa; (b) TSase enzyme assays; and (c) reagents and conditions for tryptic treatment of TSase. (b) Antisera
Rabbits were immunized with native or trypsin.treated TSase (R p H fractions) by Freund's adjuvant technic (Cohn, 1952). Three weeks following the initial injection, a single booster dose was administered, and 7 to 11 days later the rabbits were bled and the serum collected. Non-immune serum was obtained prior to immunization. The immunizing doses represented 0'7 to 1·3 mg of pure TSase protein (Garrick, 1963). The sera were heated to 56°C for 30 min prior to use. The heated sera were centrifuged at 12,000 g for 15 min at 4°C, and the clear supernatant solution was carefully separated from the sediment and surface lipid layers by means of a syringe.
(c) Immunological assay based on the specific inhibition of TSase enzyme activities We have employed four different methods which depend on an assay for TSase enzyme activity. Three of the four methods differ only in the order of additions, so that the rate constants for the various reactions involved are responsible for the observed differences. The four assays may be abbreviated as follows, with the order as written indicating the order of additions (writing E for t est enzyme, Ab for antiserum, S for su bst rat e mixture and Ag for antigen (intact enzyme, tryptic digest or cross-reacting material»: (1) inhibition of enzyme activity = E Ab S or E Ab S; (2) absorption of anti-enzyme
+
+
+
+
TRYPSIN -TREATED TRYPTOPHAN SYNTHETASE. II
85
activity = Ab + Ag + E + S; (3) competition for anti-enzyme activity = E + Ag + Ab + S; and (4) displacement of active enzyme from an enzyme/an t i-enzyme complex = Ab + E + Ag + S. Only the concentration of the component in italics is varied. There was a 5-min incubation at 4°C between each addition, excep t between Ag and S for the displacement assay, where the incubation period was deliberately varied .
(1) Inhibition of enzyme activity Anti-enzyme or anti-digest serum (diluted in a solution of 1 rng /ml. bovine plasma albumin in 0·1 M-potassium phosphate] buffer, pH 6·2 or 7·8 dep ending on the enzyme assay conditions) and a sample of enzyme (diluted in a similar fashion) were mixed at 4°C. A substrate mixture was then added, and the assay tube placed at 37°C and assayed as by Garrick & Suskind (1964). Neutralization values are based on the difference in enzyme activity in the presence of equivalent concentrations of antiserum and non-immune serum. One anti-enzyme unit is defined as the amount of antibody which will inhibit one TSu under st anda rd conditions (Suskind, 1957).
(2) Absorption of anti·enzyme activity The method used is essentially that of Suskind (1957). This method was also employed in determining the recovery of 'I'Sase-related antigen during fractionation procedures or supernatant fluid analyses in quantitative precipitin determinations. A standard antienzyme absorption curve was prepared with the original input trypsin.treated TSase as antigen. The quantity of anti-enzyme absorbed by the antigen in a given assay was determined. The recovery of antigen was then calculated by comparison to the standard curve, assuming that tryptic treatment of one TSu results in the formation of one antigen unit.
(3) Competition for anti·enzyme activity Increasing quantities of antigen were added at 4°C to a series of tubes, each of which contained an identical amount of test enzyme (4,0 to 5·0 TSu in various experiments). Subsequently, to each tube an identical quantity of antibody (2'5 to 4'0 anti-enzyme units in various experiments) was added, followed by the addition of substrate. Residual reaction 2 activity was then determined. The TSu ultimately assayed are a measure of the efficiency of enzymically inactive (or less active) antigen in competition with native TSase for anti-enzyme.
(4) Displacement of active enzyme from an enzyme anti-enzyme complex To a series of tubes at 4°C, each containing about 3 anti-enzyme units, approximately 4·5 TSu of test enzyme were added, followed by a quantity of a tryptic digest of TSase that retained about 0·5 of its original 50 TSu. After selected intervals at 4°C, substrate mixture was added. The tubes were then placed at 37°C, and reaction 2 activity was determined. The increase in TSu detected during the period of incubation at 4°C is a measure of the effectiveness of the digest in displacing active TSase from the initial enzyme/anti-enzyme complex.
(d) Quantitative precipitin tests Precipitin reactions were carried out at 4°C for up to 144 hr. The antigen/antibody precipitates were centrifuged at 4°C, washed twice with 0·1 M-potassium phosphate buffer, pH 7'8, and redissolved in 0·25 N-acetic acid. The protein in the precipitates was deter. mined spectrophotometrically at 277 mp' (Gitlin, 1949). The supernatant solutions were tested for antigen and antibody excess as follows: (1) quantitative determination of super. natant enzyme activities (Garrick & Suskind, 1964); (2) quantitative determination of antigen activity by anti-enzyme absorption, using the curve-fitting procedure described
t The potassium phosphate buffer used was a mixture of mono- and di-baaic phosphates depending on the pH required,
86
M. D. GARRICK AND S. R. SUSKIND
in section (c)2 for digest as antigen; (3) quantitative determination of supernatant solution anti-enzyme activity as in section cl; (4) qualitative testing for supernatant solution antigen and antibody by a capillary precipitin method (Kabat & Mayer, 1961).
(e) Double diffUBion in agar A modification of the Ouchterlony technio was used (Oudin, 1952). The gel medium contained 1'5% purified agar, 0'9% sodium chloride, 0'02% methiolate and 0'002% methyl orange. Special Petri plates and agar cutters were purchased from Consolidated Laboratories, Inc. (Chicago, Ill.),
3. Results (a) Inhibition of TSaae enyzme reactions
The anti- digest sera had normal levels of antd-reaction 2 activity. Anti-digest could be distinguished, however, from anti-TSase by the inhibition of TSase reaction 3F. The inhibition of TSase reactions 1 and 3F has been presented in Fig. 1. The degree of inhibition of reactions 1 and 2 is proportional to the quantity of serum added for all three sera except when AblE approaches 1·00. For almost the entire range 100 (b)
(0)
A
A~
~A
•
A
A
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'"
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lJ
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'"
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'.
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(d)
A
~
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o
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3
FIG. 1. Inhibition of TSase reactions 1, 2 and 3F by specific anti.sera. The enzyme assays are described in Garrick & Suskind (1964); the prepartion of anti-sera, in Methods and Materials, part (a), and the assay procedure, in Methods and Materials, part (b)l. % E indicates the percentage of the total input enzyme activity detected in the presence of antibody. No significant inhibition or activation of reactions 1, 2 and 3F was detected with equivalent quantities of non-immune sera. AblE indicates the ratio of antibody to TSase (antienzyme units per TSu based on previous assays of the TSase and the antisera). (a) and (b) depict the results for anti-T'Sase; (c) and (d), for one anti-digeat serum; and (e) and (f), for another anti.digest serum. (a), (c) and (e): (0) reaction 2 assays at pH 7'8; (X) reaction 2 assays at pH 6·2. (b), (d) and (f): (.) reaction 1 assays, (fl.) reaction 3F assays.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
87
AblE = 0 to AblE = 1·00,the same proportionality is found for inhibition of reaction 3F by anti-TSase (Fig. l(b». This result confirms earlier observations cited by Yanofsky (1960) and Garrick & Suskind (1963). In contrast, the anti-digest sera are less effective as inhibitors of reaction 3F (Fig. I(d) and (fl). Specific inhibition of reaction 3R by all antisera is very slight. Therefore, it is not surprising that considerable reaction 3R activity is detected in a TSase/anti-TSase specific precipitate. The lack of proportionality noted for inhibition of reaction 2 when AblE approaches 1·00 (Fig. 1(80), (c) and (e» may be accounted for by the disassociation constant of the enzyme/anti-enzyme complex. Using a modification of the method of Samuels (1963), binding constants of 0·25 to 1·10 TSu/ml. have been calculated for three sera (Fig. 2). Based on a value of 120,000 for the molecular weight of TSase and a value of 9
,
4
/)//
:J V1
t:
•
l.LJ
/
.ff:
2
~ 0
5
~1l.LJ L,j 4
3
2
o
2
4
Er-E CTSu)
FIG. 2. Equilibrium constant for the reaction of 'I'Sase with antibody prepared against native or trypsin-treated TSase. (e) results for anti-T'Sase; (., "-) two different anti-digest sera. In the insert, the results are plotted as TSase activity detected (E) ver8U8 TSase activity added (Ex) in the presence of a constant quantity of antisera. In the large Figure the results have been plotted after the theory of Samuels (1963), and values approximating the predicted linearity are obtained. The slopes of the three curves in the large :figure are - 8/TSu; therefore, K = 1/8 TSu.
M. D. GARRICK AND S. R. SUSKIND
88
2200 TSu/mg for the specific activity of pure enzyme, the equilibrium constant for the inhibition of reaction 2 of native TSase by either anti-T'Sase or anti-digest is about 10- 8 . 4 to 10- 9 ' 0 M. (b) Absorption of anti-enzyme activity by trypsin-treated TSase
The general effect of tryptic treatment on the ability of TSase to absorb antienzyme activity is illustrated in Fig. 3. Increasing quantities of native TSase neutralize proportional quantities of anti-enzyme (even in the presence of trypsin and soybean inhibitor). In direct contrast are the results for anti-enzyme absorption by trypsin-treated TSase, where a lack of proportionality is observed. Three approaches I
I
..., (/)
'c::> l1J
E
~2 c:
l1J
I .;:;
c:
-c
a
I
,
0·01
FIG. 3. A comparison of the reactions of native and trypsin-treated TSase with anti-TSase. Preparations used as antigens were taken at the times indicated by the arrows in Fig. l(a) of Garrick & Suskind (1964). The details of the assay procedure and the preparation of the antiTSase will be found in Methods and Materials, parts (b)2 and (a), respectively. 3·8 anti-enzyme units and 4·9 test TSu were used per tube. (A) TSase control; (.) tryptic digest of TSase (t = 180 min). (Redrawn from Garrick & Suskind, 1963.)
have been used to examine the basis for this non-linearity (Figs 4 and 5, Table 2). The results obtained are consistent with the hypothesis that a single new blocking antigen is formed from TSase as a result of tryptic treatment. Furthermore, the antidigest sera may be distinguished from each other and anti-TSase by their rates and extents of reaction with trypsin-treated TSase. (1) Oomparison of a reconstituted mixture of native and trypsin-treated 'I'Sase with partially inactivated trypsin-treated TSase
Trypsin-treated TSase which retains 1% of its original reaction 2 activity fractionates as though all surviving enzymic activities (Garrick & Suskind, 1964) and the
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
89
Il-ntigenic activity are associated with a single protein. In addition, the kinetics of inactivation indicate that the loss of reaction 2 activity very likely results from the hydrolysis of a single peptide bond. Consequently, it is probable that trypsin-treated TSase retaining 51 % of its original reaction 2 activity would contain a digested protein antigenically equivalent to a 50/50 mixture of native TSase and a digest which retains 1% of its original reaction 2 activity. As seen in Fig. 4, this is the case and the results are compatible with the suggestion that the reconstituted mixture and the partially inactivated TSase contain very similar antigens.
• ~o
. 2
...,
II>
'c::>
. . E
s-,
N
c
I '';:;
c
-c
.~
~~:~
. . ------0---\ • '-0
\
\. :~
0'------'-
0·02
I
0·04
MI. of Ag Fro. 4. Comparison of the anti-enzyme absorption properties of partially inactivated TSase and of a mixture of native TSas6 and trypsin-treated TSase. The antigen assay procedure in Methods and Materials, part (b)2, was used. (.) native 'I'Sase control with trypsin and soybean inhibitor present; (0) control tryptic digest of 'I'Sase retaining 1 % of its original reaction 2 enzymic activity; (.) 50/50 mixture of the native TSase and the digest; (0) trypsin.treated TSase retaining 51% of' the original reaction 2 enzymic action.
(2) Kinetics of tryptic alteration of the antigenic properties of 'I'Saee
The results in Fig. 4 suggest that hydrolysis of the peptide bond which inactivates TSase reaction 2 enzymic activity is responsible for the formation of a single antigen with altered antibody-combining properties. In Table 2 the rate of formation of the altered antigen is compared to the rate of tryptic inactivation of reaction 2. The criterion arbitrarily chosen as a measure of the antigenic alteration was the quantity of antigen required to absorb all the anti-enzyme (3,0 units) present in the assay tube. If twice as much digest (relative to native TSase) is required, one may say that it retains 50% of the antigenic activity; if ten times as much, 10%. Since the quantity of trypsin-treated TSase required to absorb all the anti-enzyme is a function of the
90
M. D. GARRICK AND S. R. SUSKIND
quantity of anti-enzyme used, the results can be considered to show that the kinetics for tryptic alteration of TSase antigenic activity are also exponential. TABLE
2
A comparison of the rates of tryptic inactivation of TSaBe reaction 2 enzymic activity and the formation of TSaBe-related antigen Relativet reaction 2 activity
Relativet antigen activity
Square root oft relative reaction 2 activity
1·00 0·37 0·25 0·12 0·083 0·032 0·012 0·010
1·00 0·72 0·48 0·43 0·30 0·19 0·11 0·057
1·00 0·61 0·50 0·35 0·29 0·18 0·11 0·10
The results of several experiments are summarized. In each experiment a TSase R 2 5 _ 31 fraction was incubated with trypsin, tryptic action was halted with soybean inhibitor at a selected time (Garrick & Suskind, 1964), and the reaction 2 activity and antigenic activity were determined. t Relative reaction 2 activity was calculated as the ratio of surviving TSu to input TSu. The square root was selected arbitrarily to emphasize the relationship between the relative reaction 2 activity and the antigenic activity. t Relative antigen activity is the ratio of the quantity of native TSase required to absorb fully the anti-TSase to the quantity of trypsin-treated TSase required to absorb fully the anti. TSase. These quantities were determined from plots resembling Figs 3 and 4.
(3) TirM-dependence of the absorption reaction
In Fig. 5 the rates of reaction of the antisera with native and trypsin-treated TSase are compared employing the absorption assay. There appears to be no increase over a period of 72 hours in the extent of reaction of native TSase with the three sera. In contrast, as reported earlier (Garrick & Suskind, 1963), the digest anti-TSase reaction is time-dependent, reaching its maximum in about 72 hours. It should be noted that the three sera employed could also be distinguished from one another by the rate and extent of reaction with digest. (c) Oompetition between native and trypsin-treated TSaBe for anti-enzyme activity
The CRM's from certain TSase mutants (Suskind, Wickham & Carsiotis, 1963), as well as trypsin-treated TSase (Fig. 3), exhibit non-linear anti-enzyme absorption plots. This lack of proportionality makes quantitative treatment of such results difficult. Another type of antigen assay based on competition between native and altered TSase for anti-enzyme was developed in the hope of finding a theoretical treatment of the results which would permit linear plots and more accurate evaluation of the results. For the assay (see Methods and Materials), one unit of competing antigenic activity is defined as the amount of antigen which competes equally with one TSu of native enzyme for anti-ensyme activity. Then the units of competing antigen per ml. in a given solution may be calculated as follows.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE . II
91
3
L __ll~,...J._~====::r:==~~=t!t===d*~=l/~!-.... II>
'c:>
QI
E >c
N
QI
I
....c:
-c
3
•
(c)
2
0·04 MI. of Ag FIG. 6. The time-dependence of the antigen/o.ntibody reaction of native and, trypsin-treated TS!IIle with homologous and heterologous antisera. See Methods and Materials, part (b)2, for experimental details. The order of additions was Ag + Ab + E + S. For the open symbols, the reactants were added about 6 min apart, whereas for the solid symbols there was a 72-hr interval between the addition of Ab and E. (e. 0) results with a native TSase fraction as antigen; ( .... .6.) results with trypsin·treated TSase retaining 1·1 % of its original reaction 2 enzymic activity. (a) depicts the results for anti.TSase; (b), for one anti-digest serum and (c), for another antidigest serum.
Let F
= units of competing antigen per ml, in the antigen sample.
G = TSu/ml. present in the antigen sample, Y = ml. of antigen sample, E T = total concentration of test enzyme (TSu). E = concentration of enzyme detected (TSu), Eo = concentration of enzyme detected with no competing antigen present (TSu).
92
M. D. GARRICK AND S. R. SUSKIND
Then the enzyme detected, E, is (active enzyme) X (fraction of total antigen which is uncombined with anti-enzyme); or,
E
=
(ET
+ GY)(Eo + FY)/(ET + FY).
(1)
This equation is not readily converted into a linear form, but if the antigen has no detectable reaction 2 activity, G = 0, and it becomes: (2)
Rearranging: (3)
Hence, a plot of (E - Eo)/Y verSU8 E - Eo should be linear, with F calculable from either the slope or the intercept. Such plots are linear (Garrick & Suskind, unpublished results) with either td2 CRM, which gives a linear anti-enzyme absorption plot, or td7 CRM, which gives a non-linear anti-enzyme absorption plot. For tryptic digests of TSase, the derivation can be revised as follows. Let J = units of competing antigen per original TSu in the digest sample, g = TSu detected per original TSu in the digest sample, i.e, fractional survival of reaction 2 activity, y = original TSu of digest prior to tryptic treatment. Prior to digestion, one antigen unit equals one TSu, by definition; therefore, the fractional survival of antigen activity, and
E = (E T
+ gy)(Eo + Jy)/(E T + Jy).
J is (I')
It should be possible to neglect g in tryptic digests, where g is small, providing J is of larger magnitude, so, (2')
and rearranging as before (3')
A plot of (E - Eo)/y versus E - Eo for trypsin-treated TSase is given in Fig. 6. The results show that J = 0·53 and g = 0'014, justifying the approximation made in going from equation (I') to (2'). Using this method, determinations with anti-TSase sera suggest that 20 to 50% of the antigenic activity has been retained in different digest preparations with g = 0·005 to 0·02. Similar determinations with one anti-digest serum give values of 40 to 60% for antigenic survival; and for another, 80 to 120%. (d) Displacement oj active TSase from a TSase/anti-TSase complex
It has been observed that trypsin-treated TSase competes effectively with native TSase for anti-enzyme activity (part C) and that there is an experimentally detectable equilibrium for the TSase/anti-TSase reaction (part (a)). Therefore, trypsin-treated TSase should displace native TSase from an antigen/antibody complex and release reaction 2 activity. Table 3 shows that a sufficient excess of trypsin-treated TSase frees at least 30% of the native TSase that had been previously inhibited by antiTSase.
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
93
4
o
3
1!------~~---____;~----~\_r_----~~ 0·05 0·20 o
0·5 (b)
0·4
fl~ 0·2
0·\
o
2
4
E-E o (TSu)
FIG. 6. Competition between native and trypsin-treated 'I'Sase for anti-TSase. For experimental details see Methods and Materials, part (b)3. In (a) the experimental points are plotted directly as 'I'Sase reaction 2 enzymic activity detected ver81Ul ml, of the antigen, trypsin-treated TSase retaining 1·4% of its original reaction 2 activity (250 'I'Sujml.), The smooth curve was calculated assuming that the trypsin-treated TSase retained 50% of its original capacity to compete for anti-enzyme, In (b) the results have been recalculated according to equation (3') in the text. The limits have been placed assuming the error of the reaction 2 assay is ± 0·2 TSu. Both the intercept and the slope indicate that slightly more than 50% of the competing antigenic activity survived digestion. The intercept value is 0'36, therefore f = 0·52; the slope is - 0·116 and therefore J= 0-53.
94
M. D. GARRICK AND S. R. SUSKIND TABLE
3
Displacement by trypsin-treated TSase of active TSase from a TSase/anti-TSase complex Addition
Controls E E+Ab (Ag +Ab) + E iE +Ag Experimental (E +Ab) +Ag Controls E E +Ab (Ag +Ab) +E Experimental (E +Ab) +Ag
0
0·2
4·42 1·57 4·30 2·95
4·32 1·58 4·29 2·97
1·0
2·31
Time in hours 4·0 6·5
28
54
TSu detected 4·34 4'39 1·08 1-13 4·26 4·24 2·97 2·68
4·03 0·97 3·71 1·32
2·71
2·76
3·08
127
73·5
4·50 1·41 4·50
4·25 1·44 4·58
4'36
4·31
4·31
4·25
4·17
3·97
1·91
2·50
2·47
2·81
2·92
For experimental details see Methods and Materials part (b}4. The results of two separate experiments are summarized. E = native TSase, Ab = anti-TSase, Ag = trypsin-treated TSase (in each experiment, original reaction 2 activity before digestion was 50 TSu). The zero-time values are based on prior assay of the antibody, test enzyme and digest. In each of the controls, the TSu detected remains the same or slowly declines during storage at 4°C. In contrast, the reaction 2 activity in the experimental tubes increases considerably before declining, indicating that active TSase has been displaced from an antigen/antibody complex. TABLE
4
Amount of precipitin for native TSase/anti-TSase and trypsin.treated TSase/anti.TSase incubated at 4°0 for 144 hr Protein Native Anti· precipiTSase enzyme tated (TSu) units (p.g) 10 20 30 40 50 60 70 80 90 100 110 120 130 150 200 50 150
67 67 67 67 67 67 67 67 67 67 67 67 67 67 67
49 90 136 158 192 198 178 112 111 85 82 77 46 50 41 o (non- 5 Oim12 mune)
Supernatant antigen Units for TSase reaction 3F t 3Rt 2(TSu) 1 0 0 0 0 0 0 0·04 0·09 0·18 0·54 0·60 0·65 0·80 HO 1·70 0·75 2·30
0 0 0 0 0 0 4 7 15 25 40 46 59 63 90 40 95
0 0 0 0 0 0 1 5 9 27 33 38 56 70 130 51 150
0 0 0 0 0 0 1 2 6 20 19 20 22 27 38 12 46
Units Capilof lary antigen test 0 0 0 0 0 0 5 10 15 36 38 40 54 83 120 60 140
0 0 0 0 0 0 0 0 0
± + + + ++ ++ + ++
Supernatant antibody Anti- Capilenzyme lary test units 44 19 9 3 0 0 0 0 0 0 0
+ + 0 0 0 0 0 0 0 0 0 0 0 0 0
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II TABLE
Trypsin Protein treated Antd- precipienzyme TSase tated (original units (,.g) TSu) 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67
37 74 107 119 96 127 48 74 66 71 62 60 61 64 57 o (non- 7 O(im- 13 mune)
10 20 30 40 50 60 70 80 90 100 110 120 130 150 200 50 150
4 (cont.)
Supernatant antigen Units for TSase reaction 3Ft 3Rt 0 0 0 0 0 0 0·04 0·05 0·06 0·10 0·11 0·15 0·19 0·26 0·34 0·15 0·45
1 2 3 4 5 5 7 12 17 19 20 28 36 38 49 18 53
95
Units of antigen 0 0 0 0 0 0 5 7 14 29 33 48 54 82 120 64 160
Oapillary test 0 0 0 0 0 0 0 0 0
+ + + + ++ ++ + ++
Supernatant antibody AntiCapilenzyme lary units test 40 21 9 8 5 5 4 4 2 4 2 3 3 4 2
+ +0 0 0 0 0 0 0 0 0 0 0 0 0
See Methods and Materials, part (d), for experimental details. A TSase R 2 8 _ 31 fraction was used for both the native and trypsin-treated TSase. The latter retained 0·8% of its original activity for reactions 1 and 2, 20% of its original reaction 3F activity and 54% of its original reaction 3R activity. All values are the mean of at least two determinations. t To increase the sensitivity of the reaction 3F assay, the reaction mixture was incubated for 5 hr at 37°C instead of the usual 1 hr (Garrick & Suskind, 1964). t Assayed at pH 6·2.
(e)
Quantitative precipitin analysisof native and trypsin-treated TSase
Quantitative precipitin observations for the homologous (native TSase) and heterologous (trypsin-treated TSase) reactions with anti-TSaso are summarized in Table 4 and Fig. 7. Although there is considerably less precipitate in the antibody excess and equivalence zones of the heterologous system, the equivalence zone for each reaction is 60 to 70 original TSu on the basis of maximum precipitation and analyses of supernatant solution. Nevertheless, it should be noted that with trypsin-treated TSase as antigen, reaction 3R activity is detected even in supernatant solutions containing excess antibody and small quantities of anti-enzyme activity are observed in supernatant solutions containing excess antigen. In quantitative precipitin deter. minations with the trypsin-treated TSase/anti-TSase'reaction, considerable variation in the maximum quantity of protein precipitated has been observed, depending on the serum used and the reaction time at 4°0. These observations are compatible with the survival of most TSase antigenic sites, but suggest that trypsin has altered the combining properties with antibody. A structural modification of the enzyme, resulting in reduced precipitability of the antigen.antibody complexes, would account for the reaction 3R activity which remains in the supernatant solutions of the antibodyexcess region as well as the anti- TSase found in the region of antigen-excess.
96
M. D. GARRICK AND S. R. SUSKIND 200
...., ....,o <1l
'0..
'u ~
o,
c:
,OJ
"2o,
100
v ;;:
'u <1l
o,
V1
o
OJ
::..
o
50
100
o
150
...
_---200
Ag (original TSu)
FIG. 7. Quantitative precipitin results for the native TSaBe/anti-TSase and trypsin-treated TSase/anti.TSaBe system. See Table 4 for the results in tabular form. (0) native TSase/anti-TSaBe system; (.) brypsln-teeated TSase/anti-TSaBe system. (0) theoretical plot for the trypsin-treated 'I'Saeejanti-T'Saee system, The theoretical points were calculated from the data in Table 4 as (quantity of specific precipitate for the native TSase/anti-TSaBe system) X (% of reaction 3R activity precipitated for the trypain-treeted TSase!anti-TSaBe syBtem)/(% of reaction 3R activity precipitated for the native TSasejanti-TSaBe system),
Maximum precipitation of one TSasp/anti.digest system is about 60% of the value obtained with the 'I'Sasefanti-T'Saee system when the two are compared on the basis of equivalent anti-enzyme units. In spite of the fact that the anti-digest system is slightly contaminated by additional antigen-antibody reactions, the diminished quantity of precipitate indicates that the two sera do not possess identical specificities for TSase. (f) Agar double-diffusion analY8is
Analyses by double diffusion in agar are depicted in Plate 1. Plate I(A) demonstrates loss of precipitability as a result of tryptic treatment of TSase. Nevertheless, the antigenic activity of trypsin-treated TSase can still be detected with this technic (Plate I(B», since mixing the antiserum and the digest in a single reservoir prevents the subsequent formation of a 'I'Sasejanti-TSase precipitin band. Plate 1(0) illustrates the confluent reaction of anti-TSase and anti-digest with native TSase.
4. Discussion (a) What fraction of the antigenic sites of TSase survive tryptic treatment?
In considering the above question, it is important to keep in mind a key difference between the antigenic determinations based on precipitin formation and the assays based on inhibition of TSase reaction 2. Specifically, the results which rely on inhibition of enzymic activity (in parts (a) to (d) depend primarily on the initial combination of TSase with antibody so as to inhibit TSase enzymic activity, whereas those involving precipitin determinations (in parts (e) and (f) depend on both the initial rapid formation of antigen/antibody complexes and the subsequent slower formation of larger insoluble antigen/antibody aggregates. The fraction of the precipitating antibody which specifically inhibits reaction 2 may be calculated from the quantitative
PLATE 1. Ouchterlony agar diffusion analysis of TSase, tryptic digests of TSase, anti-TSase and anti-digest in heterologous and homologous combinations. See Methods and Materials, part (d), for details of the procedure. (A) The center reservoir contained 240 anti-enzyme unitsjrnl. Reservoir 1 contained 250 'I'Sujml, of an R pH fraction (Garrick & Suskind, 1964); reservoirs 2 and 6, 250 'I'Sujrnl, of the same fraction in the presence of trypsin and soybean inhibitor; reservoir 3, the same concentration of TSase after tryptic inactivation to 23% of its original reaction 2 activity; reservoir 4, the same concentration of TSase after tryptic inactivation to 0·9% of its original reaction 2 activity; and reservoir 5, the same ooneentrat.ion of TSase after tryptic inactivation to 0·4% of its original reaction 2 activity. Similar results were observed for the major precipitin band with all other anti-enzyme and anti-digest sera. Additional bands were observed for all reservoirs with antidigest sera, however. (B) The center reservoir contained 120 anti-enzyme unitsjrnl. Reservoirs 1 and 4 contained 110 TSu/ml. of an R p H fraction in the presence of trypsin and soybean inhibitor; reservoirs 2 and 5, the same TSase after tryptic inactivation to 1 % of its original reaction 2 activity at a concentration that represents 280 original input TSu/mI., and reservoirs 3 and 6, the same digest as in reservoirs 2 and 5 (final concentration equivalent to 140 original input TSu/mI.), mixed with anti-enzyme (final concentration 120 anti-enzyme unit.s/rnl.). Similar results were observed with the other sera. (0) The center reservoir contained 120 'I'Sujrnl, of an R p H fraction. Reservoir 2 contained 270 anti-enzyme unitsjml. of one anti-digest; reservoir 4, 350 anti-enzyme unitejrnl, of another anti-digest; and reservoir 5, 500 anti-enzyme unitsjml. of anti-TSase. The other-three reservoirs contained other sera that do not concern us here.
[facing p. 96
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
97
precipitin observations, assuming that the neutralizing antibody possesses two identical combining sites and that each molecule of enzyme possesses one active site totally inhibitable after combination with antibody. Based on values of 160,000 for the molecular weight of the antibody, of 120,000 for the molecular weight of TSase, and of 2200 TSu/mg for the specific activity of pure TSa se, the 30 to 33 fLg of TSase in the equivalence precipitate combines with 20 to 22 fLg of neutralizing ant ibody and 160 to 170 p.g of precipitating antibody. Hence, only about 13% of the precipitating anti. TSase specifically inhibits TSase. For different determinations on various sera, values of 12 to 25% have been obtained. For comparison, Samuels (1963) finds that 15% of rabbit anti-creatine kinase is neutralizing antibody. Thus, when considering the a.ssays in the Results, parts (b) to (d), it should be kept in mind that these observations probably depend on the presence of no more than one-quarter of the antibody population. The several types of results presented in this paper cannot be accounted for unless a sizable fraction of the antigenic sites survives tryptic treatment. The simplest way to account for all the immunochemical results is to assume that all the antigenic sites are retained, and that the apparent discrepancies measure alterations in the antibodycombining properties of TSase. (b) What is the size oj the antigen present in trypsin-treated TSase?
The gel fractionations described in Garrick & Suskind (1964) were also monitored for antigenic activity by the absorption assay (Methods and Materials , part (b)2). The profile for antigenic activity was identical to the profiles for the surviving enzymic reactions on Sephadex G-75, G-I00 and G-200. For this reason it is concluded that the molecular weight of the antigenically active residual protein of t rypsin-t reat ed TSase is about the same size as the native enzyme (molecular weight = 120,000 (Carsiotis , Appella & Suskind, manuscript in preparation)). Precipitin results reported earlier (Garrick & Suskind, 1963) suggested that tryptic digests of TSase retain only 50 to 80% of th e initial antigenic activity of the enzyme and that the precipitating antigen in the digest had a maximum molecular weight of about 30,000. As initially emphasized, the survival value and the molecular weight limit were calculated on the assumption that aU of the antigen and antibody were precipitated at equivalence. The additional supernatant solution analyses in Table 4 indicate that this assumption is not justified and that the earlier interpretation of the results is invalid. (c) Is trypsin-treated 'I'Saee a successJul immunizing antigen?
Trypsin-treated TSase elicits the formation of antibody in rabbits. Because the immunizing antigen still retained 1·5 to 1·9% of the original reaction 2 activity (= 30 to 68 TSu), the possibility remains that this residual activity, representing a maximum of 13 to 31 fLg of native TSa se, was the actual immunizing antigen. Two lines of evidence rule out this possibility. First, it has been argued (Garrick & Suskind, 1964) that the residual TSase reaction 2 activity after about 99% inactivation by trypsin is not a property of the intact molecule . Second, several results in this paper indicate that the anti-digest sera are not identicalin specificity to the anti-TSase sera: (1) one anti-digest serum may be readily distinguished from anti·TSase by means of the competition assay (Results, section (cj); (2) the time-dependence observed for the extent of absorption of anti-TSase anti-enzyme activity by digest is markedly reduced (Fig. 5) when compared with the absorption of the anti-enzyme activity of the two 7
98
M. D. GARRICK AND S. R. SUSKIND
anti-digest sera; (3) when compared on the basis of anti-enzyme units, anti-TSase inhibits TSase reaction 3F enzymic activity more effectively than either anti-digest serum (Fig. 1); (4) on the basis of JLg protein precipitated per anti-enzyme unit, one anti-digest serum contains less precipitating antibody than the anti-TSase sera. Therefore, trypsin-treated TSase induces the formation of antibodies which are similar, but not identical, to anti-TSase. (d) How does trypsin modify TSase antigenic properties?
It is likely that most of the differences between the reaction of anti-TSase with trypsin-treated TSase and the reaction with native TSase are caused by a somewhat reduced affinity constant and a defect in formation of antigen/antibody aggregates. The trypsin-induced reduction in affinity constant of antigen for antibody is invoked to account for the competition results. Such an alteration combined with a reduced precipitability of antigen/antibody complexes would account for the antibody analyses of supernatant solutions in Table 4. The best evidence for a defect in the aggregation of initial antigen/antibody complexes comes from the precipitin results, where it may be seen that less precipitate is found with the trypsin-treated 'I'Sasejanti-T'Sase system than with the native TSase/anti-TSase system. In addition, the lack of an agar diffusion precipitin band for the trypsin-treated TSase/anti-TSase reaction may also be attributed to such a. defect. It is likely that the time-dependent increase in extent of the digest/anti-TSase reaction recorded in Fig. 5 is also a result of the slow formation of digest/anti-TSase aggregates. Another trypsin-induced alteration in the antigenic properties of TSase is the nonlinearity of anti-enzyme absorption (Fig. 3). This modification is not readily accounted for by either an "alteration in affinity constant or a defect in antigen/antibody aggregation. The non-linearity is not due wholly to a trypsin-induced alteration in the affinity constant for the enzyme/anti-enzyme reaction, since the lack of proportionality is also observed (Fig. 5) for a serum where competition assays detect no change in this constant. In addition, it is unlikely that the lack of proportionality is due wholly to a defect in antigen/antibody aggregation, because a non-linear absorption plot is found when digest has been incubated with anti-TSase for 72 hours (Fig. 5) or longer. This lack of proportionality in the absorption plot for trypsin-treated TSase is of special interest since the mutationally altered TSase of many TSase mutants (e.g. td 7 and td 24 ) also exhibit this property (Suskind et al., 1963). Recent unpublished evidence from this laboratory suggests that N. crassa TSase is composed of several subunits. Perhaps a trypsin-induced alteration in quaternary structure is responsible for the non-linearity of the absorption plot. It seems likely that tryptic treatment of TSase could prove very useful in the study ofimmunological properties ofthe CRM's and CRM-Iess extracts (Suskind et al., 1963). In particular, trypsin-treated TSase may contain materials which would serve as models for the "blocking" activity of certain CRM-less mutants or for the CRM's giving non-linear anti-enzyme absorption plots. (e) The 'I'Sase system for immunochemical studies
(1) It has been suggested that no more than one-quarter of the precipitating antibodies for TSase inhibit reaction 2. This result is very encouraging in the search for TSase-related materials in the CRM-negative TSase mutants, since it demonstrates
TRYPSIN-TREATED TRYPTOPHAN SYNTHETASE. II
99
that the absence of CRM may not rule out the presence of up to about 75% of the TSase antigenic sites. (2) Reactions 2, 3F and 3R do not exhibit identical antibody inhibition plots after initial reaction with antisera. Results for reaction 2 may be used for accurate quantitative determination of the enzyme/anti-enzyme reaction, since this activity can be fully inhibited. Because reaction 3R is not completely inhibited, TSase activity in antigen/antibody precipitates may be determined. (3) It is possible to measure the reaction of TSase-related antigen with antibody by assaying for several different enzymic activities, in addition to employing conventional immune-chemical techniques. Therefore, heterologous and homologous combinations of native, trypsin-treated and mutationally altered TSase with respective antisera might also serve usefully in studying antigen/antibody reactions in general, i.e. the nature of the precipitin reaction, both in quantitative precipitin analyses and during diffusion in agar. (4) The equilibrium constant for disassociation of the TSase/anti-TSase complex involved in the specific inhibition of TSase reaction 2 enzymic activity has been determined as 10- 8·4 to 10- 9·0 M. This is one of the few available determinations for a protein/anti-protein reaction. This investigation was performed while one of us (M.D.G.) was a predoctoral fellow of the U.S. Public Health Service. The research was supported by grant number C·03080 of the National Cancer Institute, National Institutes of Health. This paper is contribution number 411 of the McCollum-Pratt Institute. REFERENCES Cohn, M. (1952). Methods Med. Res. 5, 268. Garrick, M. D. (1963). Ph.D. Thesis. The .Iohna Hopkins University, Baltimore, Maryland. Garrick, M. D. & Suskind, S. R. (1963). Ann. N.Y. Acad. Sci. 103, 793. Garrick, M. D. & Suskind, S.,R. (1964). J. Mol. Biol. 9, 70. Gitlin, D. (1949). J. Immunol, 62, 437. Kabat, E. A. & Mayer, M. M. (1961). Experimentalimmunochemistry. Springfield, Illinois: Charles C. Thomas. Oudin, .T. (1952). Methods Med. Res. 5, 858. Samuels, A. (1963). Ann. N.Y. Acad. Sci. 103, 858. Suskind, S. R. (1957). J. Bact. 74, 308. Suskind, S. R., Wickham, M. L. & Carsiobis, M. (1963). Ann. N.Y. Acad. Sci. 103. 1106. Yanofsky, C. (1960). Bact. Rev. 24, 221.