Inactivation of the Plasma Protease Inhibitor aa-Macroglobulin by the Antitumor Drug cis-Dichlorodiamineplatinum(I1) *

The plasma protease inhibitor a2-macroglobulin (a2M) was reacted in vitro with cis-dichlorodiaminepla-tinum(I1) (cis-DDP). Following the reaction, a2M dem- onstrated a significantly decreased ability to bind trypsin as determined by esterase activity assays in the presence of soybean trypsin inhibitor and studies with radiolabeled trypsin. Inactivation of a2M by cis-DDP was not associated with a conversion to the “fast” electrophoretic form, as determined on nondenaturing gels, in contrast to the inactivation of azM by proteases and certain amine salts. The extent of reaction increased with the elevation of temperature within the thermal stability range of the protein; however, varia-tion of pH within the range 6.82-8.55 had little effect. Binding of [‘*C]methylamine to azM was not affected by cis-DDP. The conformational change, however, which normally accompanies this reaction did not occur. It is concluded that the aZM thiolesters are most likely not reactive sites for cis-DDP. cis-DDP-treated azM failed to dissociate into quarter subunits under denaturing and reducing conditions, suggesting cross-linking of subunits. This cross-linking may be responsible for locking the a2M quarternary structure into the “slow conformation.” tific Products AW14-120 The radiolabeled prepa- ration 60% Protein Actiuity Studies-The activity of ~ z M was studied as its ability to bind radiolabeled trypsin. Standardized quantities of protease inhibitor were reacted with cis-DDP for 3 h at 37 "C in 10 mM Tris-HC1, 90 m~ NaCI, pH 7.4. Following incubations, all samples were reacted with "'1-trypsin for 5 min and then chromatographed on Sephadex (3-150. Two peaks of radioactivity were detected. The first contained labeled trypsin associated with azM and the second, free trypsin. Binding of trypsin to azM was calculated from the fraction of radioactivity eluting in the first peak. The ability of aZM to bind trypsin following incubation with cis-DDP was also studied using the method of Ganrot (30). Following incubation of a2M with cis-DDP, each sample was reacted with trypsin at a 5-fold molar excess to the a2M for 5 min. SBTI was then added for 10 min at a 5-fold molar excess to the trypsin. The initial azM concentration was approximately 0.5 pM in all experiments. Preparations were reacted with the substrate BAPNA for 1 h at 37 "C. A4,0 ",,, was then measured. The validity of the BAPNA assay was evaluated with a series of experiments controlling for possible interaction between cis-DDP and trypsin or SBTI. Samples containing active trypsin were incubated for 15 min at 23 "C with cis-DDP at concentrations equivalent to those present in the anM assays. An identical series of samples was reacted with trypsin for 5 min and then treated with SBTI at 5-fold the trypsin concentration for 10 min. For both sets of studies, the cis-DDP used was preincubated for 2.5 h in 4.8 mM Tris-HC1, pH 7.4, at both 4.8 and 100 mM total [Cl-1, duplicating

cis-Dichlorodiamineplatinum(I1) is an uncharged square planar complex with anti-tumor activity (1)(2)(3)(4). c~s-DDP' is relatively inert when dissolved in a salt solution containing chloride at a concentration comparable to that present in the plasma (-100 mM) (5). Some degree of ligand exchange, however, is possible at the chloride sites (6). These chloride sites are probably responsible for the biological reactivity of the molecule since dissociation of the amine groups from the complex does not occur under physiological conditions (6, 7).
cis-DDP-DNA interactions are correlated with anti-tumor activity (5). It has been suggested that cis-DDP may crosslink complementary DNA strands (8). Reactions between cis-DDP and proteins have also been reported. cis-DDP inhibits the intracellular enzymes malate dehydrogenase, liver alcohol dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase when studied in vitro (9,10). Protein sulfhydryl groups react readily with cis-DDP and the presence of these groups * This work was supported by Research Grants HL-24066 from the National Heart, Lung, and Blood institute and PDT-137 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Predoctoral Fellow, Medical Scientist Training Program, National institute of General Medical Sciences (GM-07171). I The abbreviations used are: cis-DDP, cis-dichlorodiamineplatinum(I1); a2M, a2-macroglobulin; PNPGB,p-nitrophenyl-p'-guanidinobenzoate. HCI; SBTi, soybean trypsin inhibitor; BAPNA, N"benzoyl-DL-arginine-p-nitroanilide. HCI. at the active site of an enzyme may explain enzyme inactivation (10,11). By contrast, little is known about the reactivity of plasma proteins with cis-DDP. The plasma clearance of cis-DDP is biphasic, with an initial short phase of rapid clearance followed by a subsequent prolonged slow phase (12). During this second phase, between 65 and 98% of the administered cis-DDP remaining in the plasma is protein-bound.
No effort has been made to determine which plasma proteins react with cis-DDP.
a2-Macroglobulin is a plasma protein consisting of four M , = 180,000 subunits which are considered to be essentially identical (13)(14)(15). The protein reacts with numerous proteases representative of all the major protease classes (16). Reactions require active endopeptidase and are essentially irreversible. The active site of the reacting protease may, however, only be inhibited and not totally inactivated (17).
a2M is inactivated by a variety of low molecular weight ammonium analogues (18). For both protease and ammonium analogues, reactions can be followed on nondenaturing gels as an increase in mobility associated with a change in conformation in the a2M protein (18). Recent studies have demonstrated the presence of a reactive thiolester in each of the a2M subunits (19)(20)(21). Cleavage of this bond has been implicated in the covalent linkage of both amines and protease to a2M. a2M function is important in a variety of pathological conditions and disease states (22). Adams et al. (23) have suggested that a neutral protease secreted by activated macrophages has cytolytic activity against various tumors. Plasma protease inhibitors, including aZM, appear to bind and inhibit this protease (24). The presence in a2M of highly labile thiolester linkages and the possible involvement of a2M in physiological anti-tumor function prompted us to study the interaction between this protease inhibitor and cis-DDP.

EXPERIMENTAL PROCEDURES
Reagents-Trypsin, purchased from Worthington, was 70% active by active site titration with p-nitrophenyl-p'-guanidinobenzoate. HC1 (25). Soybean trypsin inhibitor, Ne-benzoyl-DL-arginine-p-nitroanilide.HC1, and PNPGB were purchased from Sigma. cis-DDP of the highest purity commercially available was purchased from Aldrich, which also supplied the reagents for metal chelation chromatography (26). ['4C]Methylamine was obtained from Amersham, I for protein iodination from New England Nuclear, and lactoperoxidase from Sigma. Electrophoresis reagents were purchased from Bio-Rad. All other reagents were of the best commercial grade available.
a& Purification-anM was purified using a modification of the procedure described by Kurecki et al. (27). A detailed description of this purification has been presented elsewhere (28). The final a2M preparation was free of contaminants as determined by NaDodS04gel electrophoresis. Densitometry scans of nondenaturing gels of the preparation indicated that greater than 90% of the a2M was in the slow conformation (18).
Radiolabeling of Proteins-a2M and trypsin were radiolabeled with using the solid state lactoperoxidase method described by David and Reisfeld (29). Radioactivity was measured using a Scien-125 12478 tific Products AW14-120 y counter. The radiolabeled trypsin preparation was approximately 60% active.
Protein Actiuity Studies-The activity of ~z M was studied as its ability to bind radiolabeled trypsin. Standardized quantities of protease inhibitor were reacted with cis-DDP for 3 h at 37 "C in 10 mM Tris-HC1, 90 m~ NaCI, pH 7.4. Following incubations, all samples were reacted with "'1-trypsin for 5 min and then chromatographed on Sephadex (3-150. Two peaks of radioactivity were detected. The first contained labeled trypsin associated with azM and the second, free trypsin. Binding of trypsin to azM was calculated from the fraction of radioactivity eluting in the first peak. The ability of aZM to bind trypsin following incubation with cis-DDP was also studied using the method of Ganrot (30). Following incubation of a2M with cis-DDP, each sample was reacted with trypsin at a 5-fold molar excess to the a2M for 5 min. SBTI was then added for 10 min at a 5-fold molar excess to the trypsin. The initial azM concentration was approximately 0.5 pM in all experiments.
Preparations were reacted with the substrate BAPNA for 1 h at 37 "C. A4,0 " , , , was then measured. The validity of the BAPNA assay was evaluated with a series of experiments controlling for possible interaction between cis-DDP and trypsin or SBTI. Samples containing active trypsin were incubated for 15 min at 23 "C with cis-DDP at concentrations equivalent to those present in the anM assays. An identical series of samples was reacted with trypsin for 5 min and then treated with SBTI at 5-fold the trypsin concentration for 10 min. For both sets of studies, the cis-DDP used was preincubated for 2.5 h in 4.8 mM Tris-HC1, pH 7.4, a t both 4.8 and 100 mM total [Cl-1, duplicating the chemical milieus for trypsin and SBTI in the agM assays. Following preparation, all samples were incubated with substrate as described above.
Polyacrylamide Gel Electrophoresis-Gel electrophoresis under nondenaturing conditions was conducted on slabs using the Tris/ borate buffer system described by Nelles et al. (31). Ammediolbuffered NaDodS04-containing gel systems with stacking and resolving levels were prepared as described by Wyckoff et al. (32). The gels were stained with Coomassie brilliant blue R-250 (CBB-R-250).
Gel Densitometry-Native gels were subjected to densitometric analysis using a Gelman ACD-15 automatic integrating densitometer with a maximum sensitivity of 0.1 absorbance. CBB-R-250-stained protein absorbed maximally at 565 nm.
Dialysis Experiments-The reversibility of the inactivation of a2M by cis-DDP was tested in the following experiment. Radiolabeled azM was added to a solution containing 1.22 mM cis-DDP, 90 mM NaCI, and 10 mM Tris-HC1 (final volume, 410 pl). Immediately following addition of the cis-DDP, a 20-4 aliquot was removed from the preparation and reacted with trypsin at 5-fold the a2M concentration for 5 min and then excess SBTI for 10 min. Remaining esterase activity was assayed with the substrate BAPNA. The initial reaction mixture was incubated for 2.5 h at 37 "C, at which time a second 20pl aliquot was removed and treated identically to the first. The remainder of the reaction medium was then dialyzed against 4 liters of 10 mM Tris-HCI, 90 mM NaCl. Additional 20-p1 aliquots were removed for repetitive analysis at 3, 12, and 30 h. a2M concentration was standardized through comparison of the radioactivity associated with each sample. Following removal of each aliquot, 4 liters of fresh dialysis buffer was substituted.

RESULTS
Trypsin and SBTI Activity in cis-DDP-Stable activity of both trypsin and SBTI under all imposed conditions is essential for the validity of the a2M Ganrot assay. The esterase activity of trypsin was measured in Table I following incubation with cis-DDP for the length of time required in the a2M assays. It has been suggested that in uiuo cis-DDP reactivity is highly dependent on chloride concentration. The reactive species forms through exchange of solvent for C1which takes place much more rapidly at cytoplasmic [Cl-] (-5 mM) than at plasma [Cl-] (-100 mM) (5,33). Since physiologic reaction between drug and anM would occur in the plasma, a comparison of the nature of the reaction at 4.8 and 100 mM [Cl-j was important. Table I demonstrates that the trypsin esterase activity associated with the free protease is not affected by the cis-DDP or chloride concentration of the medium to which the trypsin is exposed for 15 min before transfer to BAPNA assay buffer. Table I also demonstrates that the SBTI used in these assays is capable of inhibiting essentially all of the trypsin esterase activity at every tested cis-DDP and chloride concentration. a * Activity following Treatment with cis-DDP-Preparations containing identical quantities of azM were incubated with varying concentrations of cis-DDP at 37 "C for 2,4, and 8 h. Reaction mixtures were prepared in which the final [Cl-I was both 4.8 mM and 100 mM. Following incubations, a2M activity assays were performed using the Ganrot method as described under "Experimental Procedures." A dose-and time-dependent inactivation of azM in its capacity to bind trypsin with detectable esterase activity is clearly shown at both chloride concentrations (Fig. 1). In consideration of these data, 90 mM NaC1, 10 m~ Tris-HCl, pH 7.4, was adopted as a standard reaction medium for all the remaining experiments.
In a second series of experiments, a2M-trypsin complex was formed in the absence of cis-DDP. The preformed complex was then treated with cis-DDP, SBTI, and BAPNA substrate duplicating conditions present in the anM Ganrot assays. The measured esterase activity of trypsin which was already bound to a2M was unaltered by cis-DDP. This result suggests that the diminished esterase activity demonstrated by cis-DDP- Fig. 1 is a result of a decreased capacity to bind trypsin rather than a loss of esterase activity by bound trypsin.
To further test this hypothesis, cis-DDP-treated anM was reacted with '251-trypsin and then subjected to gel fiitration on Sephadex G-150. Using similar reaction conditions and gel fiitration, it has been demonstrated that as many as 4 mol of '"1-trypsin may chromatograph associated with 1 mol of a2M, although a maximum of 2 of these mol demonstrate retained esterase activity (34). Native anM which had not been treated with cis-DDP co-chromatographed with essentially 100% of the active trypsin reacted with it at a 2.1:l molar ratio for 5 min. a2M which had been treated with 1.50 m~ cis-DDP for 3 h and then trypsin for 5 min at the same molar ratio chromatographed with less than 0.9 mol of trypsin/mol of inhibitor. Treatments with 60 p~ and 600 PM cis-DDP resulted in a2M preparations which chromatographed with 1.8 and 1.4 mol of trypsin, respectively. These data demonstrate that czs-DDP treatment can decrease the ability of a2M to bind trypsin. As was expected, after cis-DDP incubation, slightly more '"I-trypsin became associated with the a2M preparation than was measurable by esterase activity assay. Sottrup-Jen-  Nondenaturing Gel Electrophoresis of cis-DDP-treated aJ4-When reacted with trypsin, the mobility of a2M on nondenaturing gels increases from "slow" to "fast" secondary to a conformational change in the asM protein (18). Fig. 2 demonstrates this change. The fast form of a2M produced by reactive ammonium salts is electrophoretically identical with that produced by proteases (18).
Samples of anM were incubated with cis-DDP at concentrations equivalent to those used in the u2M assays. Alteration in u2M electrophoretic behavior occurred only at cis-DDP concentrations greater than 1 mM after 3 h. At these concentrations, 2 faint bands of slow mobility consistently were present. These experiments were, in general, notable for the stability and preservation of the slow electrophoretic conformation of asM at cis-DDP concentrations where significant levels of inactivation were detected by the activity assay described above. In no experiment was protein inactivation correlated with conversion to the fast electrophoretic form (Fig. 2). This is in direct contrast to the result obtained with reactive amine salts (18).
The altered ability of cis-DDP-treated a2M to complex protease was then studied as reduced capacity to change electrophoretic conformation. Eight solutions containing 0.5 p~ a2M were incubated with cis-DDP at concentrations between 2.7 p~ and 1.67 mM for 3 h. All 8 samples were reacted with trypsin for 5 min at a trypsin to anM molar ratio of 5 and then excess SBTI. Nondenaturing gels of these solutions were scanned by a densitometer. Peaks were automatically resolved and fractional protein content was determined. Fig. 2 shows four of the eight samples. At a cis-DDP concentration of 6. Densitometric analysis of these experiments is shown in Fig. 3. A shoulder of mobility intermediate between the "slow" and "fast" form is first detected at 17 p~ cis-DDP. The proportion of anM electrophoresing at slower mobilities increases with increasing cis-DDP concentrations. The inability of cis-DDP-treated a2M to convert to the "fast" conformation following trypsin treatment is consistent with its failure to bind trypsin. The possibility, however, that cis-DDP affects the anM structure in a manner which interferes with conformational change cannot be excluded.
CRB-R-250 dye binding is directly proportional to the mass of protein in a band for any given protein within the concentration and mobility ranges applied to nondenaturing gels in this study (35). The relative dye binding capacities of native "slow" w~M , protease complexed "fast" arM, and unM following cis-DDP treatment were determined in a series of experiments in which equal masses of each type of protein were electrophoresed. Following gel staining, densitometric measurements of bound dye were essentially identical for each of the three asM species (data not shown). These data permitted direct interpretation of the densitometry scans in Fig. 3 as representing actual protein content in the electrophoretic bands. In Fig. 4, cis-DDP incubation concentration is plotted against the percentage of a2M which retains the capacity to bind trypsin and convert to the fast mobility conformation.
The Behavior of cis-DDP-treated anM on NaDodSOr Gels--arM was incubated with cis-DDP and applied to denaturing gels (Fig. 5). Before application, solutions were reacted with 4 mg/ml of dithiothreitol, 1% NaDodSOJ for 40 min at 37 "C. The possibility of interaction between unreacted cis-DDP and dithiothreitol was recognized. The NaDodS04 dithiothreitol incubation media, however, contained dithio-  Incubation of azM with cis-DDP, followed by reaction with trypsin and SBTI, was performed as described in Fig. 2. Peaks from gel scans were automatically resolved and integrated and the per cent protein migrating with a mobility equivalent to that of the fast conformation is plotted against initial cis-DDP incubation concentration. threitol at 16 times the cis-DDP molarity for even the sample with the highest drug content. Adequate dithiothreitol was therefore available for complete reduction of the disulfide bonds present in arM (36, 37). The effect of such reduction should be conversion of a2M to quarter subunits (13) and essentially total conversion was achieved in the absence of cis-DDP. At a cis-DDP incubat.ion concentration as low as a b c d e f g FIG. 5. NaDodS0,-gel electrophoresis of cis-DDP-treated a z M . alM was reacted with cis-DDP for 4 h at 37 "C. Samples were then incubated in NaDodS04-dithiothreitol and subjected to electrophoresis under denaturing conditions. The cis-DDP incubation concentrations for a through g were 0, 7.9 pM, 27 p~. 90 pM, 302 pM, 753 PM, and 1.88 mM, respectively. 7.9 PM, a faint hand of very slow mobility is visible. At 90 PM, a second band of even slower mobility becomes apparent. At higher drug concentrations, significant quantities of protein electrophorese in these two regions. These two bands correspond to those obtained by Harpel (37) when a2M was subjected to NaDodS04-gel electrophoresis without prior reduction and most likely represent half and whole molecules of a2M. Further evidence that these bands represent half and whole molecules is provided by comparison to the electrophoretic patterns observed by Barrett et al. (18) when asM was treated with glutaraldehyde. These data demonstrate that cis-DDP prevents dissociation of the anM quarter subunit under strongly denaturing and reducing conditions. It is highly likely that this effect of cis-DDP results from the formation of crosslinking coordinate covalent bonds between the two reactive sites on the drug and separate subunits of azM.
Two additional bands of faster mobility than the a2M quarter subunits are present in the untreated control of Fig.  5. These well described bands result from incubation of arM a t temperatures of 37 "C or above in denaturing buffer (38, 39). The diminished intensity of these bands following cis-DDP treatment parallels the decrease in protein mass electrophoresing as resolved quarter subunits.
The Effect of Temperature andpH-Identical preparations of a2M were incubated at various temperatures for 2.5 h both with and without a standard concentration of cis-DDP (Fig.  6). Activity assays using the substrate BAPNA were then performed. In the absence of drug, the activity of the native protein is relatively stable for 2.5 h over a range of increasing  temperatures to 52 "C. asM preparations treated with cis-DDP lost the capacity to protect trypsin esterase activity in a manner which was highly temperature-dependent. At 4 "C, the cis-DDP-treated aZM retained 94% of the activity of the untreated control while a t 52 "C only 1% of the relative activity was retained.
The pH dependence of the cis-DDP-a2M interaction is studied in Fig. 7. Native azM was relatively stable at 37 "C for 2.5 h throughout the tested pH range of 6.82-8.55. Significant inactivation of a2M by cis-DDP occurred a t each pH tested. Although alteration in reactivity with pH was small, reaction seemed to take place optimally near neutrality.
Kinetic Aspects of the Ability of cis-DDP-treated aJ4 to Bind Trypsin-At room temperature, binding of trypsin to a2M is rapid. The possibility that treatment of (YZM with cis-DDP results in "sluggishly" reacting aZM is explored in Fig.  8. Identical cis-DDP-treated samples of a2M were incubated with trypsin for between 2 and 60 min before addition of excess SBTI. Following 2 min of trypsin reaction time, the azM preparation demonstrated 86% less trypsin esterase activity than the untreated control. A 10-min incubation resulted in a preparation with slightly greater activity; however, nearly an 80% loss was maintained through 1 h, at which time apparent equilibrium had been reached.
Reversibility of Inactivation--azM treated with 1.25 mM cis-DDP for 2.5 h was dialyzed extensively as described under "Experimental Procedures." The ability of the a2M to bind trypsin and preserve esterase activity was tested before and after reaction with cis-DDP as well as at various stages of dialysis. Approximately an 80% loss in activity was initially observed. Up to 30 h of dialyses failed to reverse this inactivation. There remained over this time period either tightly bound platinum, inhibiting activity of many of the a2M molecules, or some degree of irreversible protein modification.
The Reactivity of the Thiolester in cis-DDP-treated a&f-Native aaM may covalently bind up to 4 mol of methylamine per mol of protein (40). The methylamine-reactive site is the labile thiolester present in each azM subunit (19,21). Reaction with methylamine induces a conformational change in a2M producing the electrophoretic "fast" form (18). a2M was incubated for 3 h in solutions containing 50 pM, 555 p~, and 1.85 mM cis-DDP. Following extensive dialysis, each preparation was reacted for 40 min with 200 mM [14C]methylamine and again dialyzed exhaustively. The binding of methylamine to untreated azM was calculated a t 3.6 mol/mol. Calculated methylamine binding ratios for ~Y Z M treated with 50 PM, 555 ,UM, and 1.85 mM cis-DDP were 3.6, 3.8, and 3.9, respectively. Treatment with cis-DDP, therefore, did not interfere with the reaction between methylamine and the asM thiolesters. This strongly suggests that cis-DDP-cYzM interaction does not involve asM thiolester bonds.
a2M was treated with different concentrations of cis-DDP for 3 h and then dialyzed thoroughly. Each solution was then made 200 mM in methylamine for 60 min before application on a nondenaturing gel. cis-DDP treatment clearly inhibits the conformational change that causes increased electrophoretic mobility in a2M following reaction with methylamine ( Fig. 9). This inhibition is cis-DDP concentration-dependent. FIG. 8. The kinetics of reaction of trypsin with azM following incubation of azM with cis-DDP. Seven equivalent preparations of asM were incubated with 1.67 m~ cis-DDP at 37 "C for 2.5 h. An eighth sample with no cis-DDP was also incubated. Following incubation, the fiist seven samples were reacted with trypsin for the length of time plotted on the abscissa. The control ( c ) containing no cis-DDP was reacted with trypsin for 2 min. All samples were then treated with SBTI for 10  and then dialyzed extensively. Each preparation was then reacted with 2(H) m.~ methylamine for 60 tnin and electrophoresecl on a nondenaturing gel except for sampit rr \vhich was treated with neither cis-I)I)t' or methylamine. This c,is-I>I)l' incubation c.onc.entrations were 0. l(H) ql. 600 1-07. and I .X5 nlM for lnnes h-r. respectively.

Inhibition
of conformational change was demonstrated in this experiment following reactions equivalent to those which resulted in the maximal incorporation of ["C]methylamine into ct2M as described above. The inability of ck2M molecules to convert to the "fast" conformation in Fig. 9 may not, therefore, be attributed to decreased binding of methylamine to c\,M as will be discussed below.
The studies presented in this paper have demonstrated altered function of the plasma protease inhibitor (tLM following incubation with cis-DDI'. This altered function was demonstrated following reaction for 3 h with 17 11.~ cis-l)l)l' in an experiment testing the ability of drug-treated ci2M to undergo conformational change following reaction with trypsin. Time course studies using the Ganrot technique (30) indicated that maximal inactivation of (tLM by cis-11111' was probably not achieved until after 8 h and definitely not by 2 h. In addition. a physical alteration in ct2M resulting from incubation with 7.9 PM cis-I)I>I' for 4 h was demonstrated using NaI>odSOIgel electrophoresis.
It is suggested that these in 1%itro data mav be representative of in t.ilw plasma chemistry occurring d&ing some of the more intensive therapeutic regimens using cis-I>DI' (2).
Reaction between ck2M and an active endopeptidase is dependent upon proteolytic cleavage of a susceptible site in +M, termed the "bait region" (16). A covalent linkage may be formed between protease and inhibitor; however. this bond is not essential for the inhibitory capacit: of ck2M (41). Conformational change within a>M, however, IS most likely essential to the completion of the irreversible reaction (34). c\?M demonstrated decreased ability to hind radiolabeled trysin following reaction with cis-I>I)I'.
This result is consistent with some form of "bait region" modification.
Alternatively this result may he explained as failure of the ct?M molecule to change conformation and trap reacting protease. Many proteins are completely unaffected by high concentrations of cGI)I)P (10). Although other amino acids ma? react with c,is-DDI', it has been suggested that sensitivity of a protein to the drug depends upon that protein having an essential sulfhydryl group at its active site (10. 11). ~I,M possesses no free sulfiydryl groups (21). The labile thiolesters present in ck2M were explored as alternative reaction sites. cis-DDE failed to inhibit reaction of the subunit thiolesters with ["'C]methvlamine.
These data are most consistent with lack of interaction between cis-11111 and the ct,M thiolesters. If, indeed, reaction occurs at these sites, it occurs without significantly altering the thiolester chemistry. This is c,onsidered unlikely.
cis-DI>I' is a bifunctional reagent in which the two active sites are relatively close together. Attempts in this laboratoq to use cis-DDP as a general cross-linking reagent for otherwise dissociable polypeptide chains have been unsuccessful for a variety of proteins.-When c12M was reacted with cis-I>I)I'. a significant fraction of the protein failed to dissociate into quarter subunits under denaturing and reducing conditions.