Biochem. J. (1982) 201, 119-128 119Printed in Great Britain

Functional modifications of a2-macroglobulin by primary amines

Kinetics of inactivation of a2-macroglobulin by methylamine, andformation of anomalous complexes with trypsin

Fred VAN LEUVEN, Jean-Jacques CASSIMAN and Herman VAN DEN BERGHEDivision ofHuman Genetics, Department ofHuman Biology, University of Leuven, Minderbroedersstraat 12,

B-3000 Leuven, Belgium

(Received 29 June 1981/Accepted 1 September 1981)

The unique steric inhibition of endopeptidases by human a2M (a2-macroglobulin) andthe inactivation of the latter by methylamine were examined in relation to each other.Progressive binding of trypsin by a2M was closely correlated with the loss of themethylamine-reactive sites in a2M: for each trypsin molecule bound, two such siteswere inactivated. The results further showed that, even at low proteinase/a2M ratios,no unaccounted loss of trypsin-binding capacity occurred. As a2M is bivalent fortrypsin binding and no trypsin bound to electrophoretic slow-form a2M was observed,this indicates that the two sites must react (bind trypsin) in rapid succession. Reactionof [1'4Clmethylamine with a2M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In thesubsequent slow phase trypsin had no such effect. These results prompted furtherstudies on the kinetics of methylamine inactivation of a2M with time of methylaminetreatment. It was found that conformational change of a2M and decrease in trypsinbinding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. Thelatter decreased rapidly, following pseudo-first-order kinetics. Conformational changewas much slower and followed complex kinetics. On the other hand, binding of 1251-labelled trypsin to a2M did follow the same kinetics as the conformational change. Thisdiscrepancy between total binding (1251 radioactivity) and trypsin-inhibitor-resistantbinding of trypsin indicated formation of anomalous complexes, in which trypsin couldstill be inhibited by soya-bean trypsin inhibitor. Further examination confirmed thatthese complexes were proteolytically active towards haemoglobin and bound 125I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition bysoya-bean trypsin inhibitor was slowed down as compared with reaction with freetrypsin. The results are discussed in relation to the subunit structure of a2M and tothe mechanism of formation of the complex.

a2-Macroglobulin (a2M) is unique among theproteinase inhibitors present in the circulation ofmammals, by virtue of its typical steric modeof inhibition of nearly all known endopeptidases(Starkey & Barrett, 1977). Only enzymically activeproteinases are bound, indicating that the proteo-lytic event (cleavage of the polypeptide chain ofa2M) (Harpel, 1977) triggers the actual formationof the a2M-proteinase complex. The complex main-tains the enzymic activity of the proteinase towardslow-molecular-weight substrates, but proteolysis is

Abbreviation used: a2M, a2-macroglobulin.

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inhibited almost completely. Analytically, the com-plexes are distinguished from native a2M by theirfaster electrophoretic mobility despite a morealkaline pI (Ohlsson & Skude, 1976; Barrett et al.,1979; Parsons & Romeo, 1980; Van Leuven et al.,1981a). Of biological relevance was the finding of acellular receptor specific for a2M-proteinase com-plexes (Debanne et al., 1975; Van Leuven et al.,1979; Kaplan & Nielsen, 1979). Recognition leadsto rapid internalization of a2M-proteinase com-plexes by a receptor-mediated endocytosis mech-anism (Van Leuven et al., 1980), similar in charac-teristics to the one proposed for low-density

0306-3275/82/010119-10$01.50/1 (© 1982 The Biochemical Society

F. Van Leuven, J.-J. Cassiman and H. Van den Berghe

lipoprotein (Goldstein et al., 1979). Rapid clearanceof a2M-proteinase complexes from the circulationin vivo (Ohlsson & Skude, 1976) is likely to bemediated by a similar mechanism.

Primary amines inactivate a2M as a proteinaseinhibitor (Steinbuch et al., 1968), by a covalentmodification of a 'pseudo-active centre' (Swenson &Howard, 1979). Treatment of a2M with proteinaseor with methylamine results in the same conforma-tional form of a2M, as judged from electrophoreticmobility, isoelectric-focusing pattern and kinetics ofreceptor-mediated endocytosis by fibroblasts inculture (Van Leuven et al., 1981 a). We have furthershown that proteinases transiently activate themethylamine-reactive site, which was interpretedto show that the methylamine-reactive site actuallyholds a2M in the native conformation and isactivated by proteolysis leading to the formation ofcomplex (Van Leuven et al., 198 lb).

The present work was undertaken to examinequantitatively the inter-relationship of trypsin andmethylamine actions on a2M. A direct correlationwas found between the trypsin-binding sites and the'methylamine-reactive' sites on the formation ofa2M-trypsin complex. Studies on the kinetics ofmethylamine incorporation were done in relation toconformational change and trypsin-binding capacityof a2M. The results show that under certain condi-tions a2M-trypsin complexes can be formed thatare proteolytically active towards haemoglobinand that bind soya-bean trypsin inhibitor to theactive site of trypsin in the complex.

ExperimentalMaterials

The Radiochemical Centre (Amersham, Bucks.,U.K.) supplied us with [14C]methylamine (specificradioactivity 61.1 mCi/mmol) and with Na'251.1 - Chloro - 4 - phenyl - 3 - tosylamidobutan - 2 - one('TPCK')-treated trypsin and soya-bean trypsininhibitor were from Worthington Biochemical Corp.(Freehold, NJ, U.S.A.). Radioautography was doneon 3H-Ultrofilm (LKB Produkter, Stockholm,Sweden).

Purification ofa2MHuman citrated plasma (haptoglobin type 1-1)

was obtained from the plasmapheresis service of thelocal blood bank and processed on the day ofdonation. In the initial stages of purification onlyplastic laboratory ware was used.

Plasma a2M was isolated after treatment of theplasma with BaCl2 and BaSO4 as described pre-viously (Van Leuven et al., 1979). Further purifica-tion steps were: precipitation of a2M with poly-ethylene glycol-6000 (precipitate between 4 and 8%,w/v) and gel filtration on Ultrogel AcA-22 (LKB)

[column dimensions 5 cm x 95 cm; eluent phosphate-buffered saline (0.15 M-NaCI/20 mM-sodium phos-phate buffer, pH 7.4)1. The final purification stepconsisted of chromatography on a column (2.6 cm x90 cm) of Blue Sepharose (Pharmacia, Uppsala,Sweden), with 25 mM-sodium phosphate buffer,pH7.7.

Quantitative determination of a2M after each stepand in column fractions was performed by 'rocket'immunoelectrophoresis (Van Leuven et al., 1978).Protein was determined by the method of Lowryet al. (1951), with bovine serum albumin as stan-dard. A typical purification procedure, starting from300ml of plasma, resulted in a preparation contain-ing 97% a2M in a yield of 58%.

Electrophoretic techniques in thin-layer polyacryl-amide gels

Electrophoretic mobility of native proteins wasexamined in 5% polyacrylamide gels. A discon-tinuous buffer system was used. Reservoir buffer(upper and lower) was 41 mM-Tris/40mM-boratebuffer, pH 8.6; stacking gel was 4% polyacrylamidein 54mM-Tris/20mM-H2SO4 buffer, pH6.1; separ-ating gel was in 95 mM-Tris/HCl buffer, pH 5.7Samples were prepared in reservoir buffer containing10% (v/v) glycerol. Gels were cast in cassettes(Pharmacia) of dimensions 8 cm x 8 cm x 0.27 cmand run for 3 h at 125 V.Sodium dodecyl sulphate /polyacrylamide - gel

electrophoresis was performed as described byLaemmli (1970) on gradient gels (4-20% poly-acrylamide), of dimensions 8cmxl4cmxO.27cm(Pharmacia). Samples were prepared in buffer con-taining 1 % (w/v) sodium dodecyl sulphate and 1 %(v/v) 2-mercaptoethanol and left for 10min at 800Cbefore application on the gel. Electrophoresis was for16h at 50V. The apparatus used was a PharmaciaGE-2/4 gel-electrophoresis tank with circulatingbuffer, operated at room temperature.

Isoelectric focusing was done on a Multiphorapparatus and the appropriate electrodes for focus-ing (LKB). Gels were commercially available andrun as recommended by the manufacturer (PAG-PLATE, pH3.5-9.5; LKB). All gels were fixed in10% (w/v) trichloroacetic acid, stained with Coo-massie Brilliant Blue (at 600C) and destained bydiffusion. After shrinking of the gels in methanol/water (1: 1, v/v) overnight, the gels were dried forradioautography. Photographs were made with aPolaroid camera with positive/negative type 665film.

Quantitative determination of radioactivity wasdone by excision of Coomassie Brilliant Blue-stainedbands with a razor-blade. The pieces of gel weredissolved in 1 ml of concentrated H202 at 560Covernight. After addition of 15ml of scintillation'cocktail' (Instagel II; Packard Instruments, La

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Grange, IL, U.S.A.), radioactivity was counted(Packard Tri-Carb liquid-scintillation counter).Counting efficiency was determined with an internalstandard.

Trvpsin activitvTrypsin enzymic activity towards benzoylarginine

p-nitroanilide was measured at 370C as describedpreviously (Van Leuven et al., 1981a): the assaymixture contained 0.1 M-Tris/HC1 buffer, pH 8.2,20mM-CaCl2, 2 mM-benzoylarginine p-nitroanilideand enzyme in a final volume of 2.5 ml. Absorbanceat 410nm was continuously recorded. Enzymicactivity towards Chromozym PL (tosylglycylprolyl-lysine p-nitroanilide; Boehringer, Mannheim,Germany) was measured under similar conditions,at a substrate concentration of 0.3mm. Activitiesare expressed as the change in A410/min at 370C.When indicated., soya-bean trypsin inhibitor wasadded, equivalent to the amount of trypsin presenton a weight basis.

Proteolytic activity was measured towards PH]-acetylated haemoglobin. Bovine haemoglobin (typeI; Sigma Chemical Co., St. Louis, MO, U.S.A.)was labelled with 13Hlacetic anhydride (3 Ci/mmol;The Radiochemical Centre): 600mg of haemo-globin was treated with 25 mCi of anhydride in0.5 M-borate/ 1.4M-sodium acetate buffer, pH 9.0, for24h at 0°C. The labelled protein was collected andfreed from anhydride that had not reacted by re-peated precipitation with trichloroacetic acid andfinally dialysed against 0.16M-NaCI. The final prep-aration contained 0.62mg of protein/ml and wasstored at -200 C. It contained 4500 c.p.m./,ug ofprotein, of which 0.4% was not precipitated bytrichloroacetic acid.

The assay mixture contained, in a final volumeof 0.5 ml, 50mM-Tris/HCI buffer, pH8.2, l00,l ofthe labelled haemoglobin preparation and eithertrypsin (1,ug) or a2M (200,ug). Incubation for 1 h at370C was followed by addition of 0.5 ml of outdatednewborn-calf serum (GIBCO, Grand Island, NY,U.S.A.) and 0.4 ml of 50% (w/v) trichloroaceticacid. After 30 min in ice the solution was cleared bycentrifugation, and 1 ml of the supernatant wasadded to 15 ml of Instagel II (Packard). Radio-activity was counted in a Tri-Carb liquid-scintillationcounter (Packard) with external standardization.

Active-site titration of bovine trypsin (1-chloro-4-phenyl-3-tosylamidobutan-2-one - treated; Wor-thington) was done with p-nitrophenyl p-guanidino-benzoate (ICN Corp., Cleveland, OH, U.S.A.) asdescribed by Chase & Shaw (1970).

Results

Effect oftr.vpsin on methvlamine incorporationIn a first series of experiments we wanted to

examine the quantitative relationship between trypsinbinding to a2M and the 'methylamine-reactive' sites.This was done by titration with ['4Clmethylamineof the remaining sites in a2M-trypsin preparationswith different degrees of complex-formation.

By titration of a2M with trypsin, a classicaltitration curve was constructed for trypsin-inhibitor-resistant trypsin activity (Fig. la). The plateau wasreached at an a2M/trypsin molar ratio of 1:3. Inthe linear part more than 95% of the trypsin addedwas not inhibited by soya-bean trypsin inhibitor.Samples of all data points, examined by polyacryl-amide-gel electrophoresis, under denaturing andreducing conditions, showed progressive binding oftrypsin to be accompanied by a decreasing amountof a2M monomer (Mr 185000) and increasedamounts of a protein band with apparent Mr 85 000(Fig. lb). Bands at Mr 127000 and 61000 are dueto thermal fragmentation of native a2M (Harpelet al., 1979). Bands at Mr 25 000, 12000 and 10 000were also seen when free trypsin was run undersimilar conditions. A small amount of undissociatedcomplexes remained in the upper part of the gel(Harpel et al., 1979).The different a2M-trypsin preparations (corre-

sponding to each data point in Fig. 1) were examinedfurther in two ways: by addition of excess of 12511labelled trypsin (Van Leuven et al., 1979) and byincubation with [14C]methylamine. In both cases,a2M was separated from excess of reagent byrate electrophoresis on 5% polyacrylamide gels,and the amount of radioactivity present in a2Mbands was determined. Untreated a2M (no trypsinadded) was used as a control. The results showed aclose parallel between ['4Clmethylamine incorpora-tion and binding of 251I-labelled trypsin over theentire titration curve. The sample at the plateau(a2M fully saturated with trypsin) did not bindappreciable amounts of 125I-labelled trypsin or of[14C]methylamine. These results confirm and extendthe finding that, after formation of complex withtrypsin, the methylamine-reactive sites in a2M are nolonger reactive.

Active-site titration of trypsin showed the prep-aration to contain between 60 and 70% activeenzyme, indicating the molar binding ratio ofactive trypsin to a2M to be about 2: 1. Incorporationof [14Clmethylamine was found to be between 3.7and 4.1 mol/mol of a2M (Mr 725 000). Thus, foreach molecule of trypsin bound, two methylamine-reactive sites were inactivated.We have shown that [14C]methylamine incorpora-

tion into a2M displayed biphasic reaction kineticswith respect to time, and that incorporation wasincreased by formation of trypsin complex (VanLeuven et al., 198 l a,b). These two phenomena wereexamined in the same experiment: [14Clmethylamineincorporation was measured as a function of time;

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121

F. Van Leuven, J.-J. Cassiman and H. Van den Berghe

>,01-o0N.Rl

g:*_m5

-185

-127

-85

-61 Px

0

20~

0 50 100 150 200 400

Trypsin (ug)

Fig. 1. Titration ofa2M with trvpsin, I251-labelled trypsin and ['4Clmethvlamine(a) Portions (2 mg) of human a2M were treated with the amounts of trypsin indicated in 25 mM-Tris/HCI buffer,pH 8.0. Samples were taken for the determination of trypsin-inhibitor-resistant amidase activity (0) and for poly-acrylamide-gel electrophoresis (b). The equivalent of 750,ug of a2M from the original incubation mixtures wasfurther treated with either excess of 1251I-labelled trypsin (for 15min at 25°C) or with 25 mM-['4Clmethylamine (for18 h at 370 C). Radioactivity in a2M was determined by excision and dissolution of stained bands after separation on5% polyacrylamide gels. The data are expressed relative to completely saturated a2M: for amidase activity (at400,ug of trypsin) activity measured was 0.026 A410 unit increase/min (for lOO,ug of a2M). Native a2M (no trypsinadded) bound respectively 31 240 c.p.m. of '251-labelled trypsin and 6035 c.p.m. of [14Clmethylamine per lO,ug ofprotein. Binding is expressed relative to these results: 0, binding of 1251I-labelled trypsin; O, [14Clmethylamineincorporation. (b) Polyacrylamide-gel electrophoresis after denaturation and reduction. Samples (taken fromtitration mixtures, see above) were treated with sodium dodecyl sulphate and 2-mercaptoethanol (1% each, finalconcentrations) for 10min at 800C. In each lane 20,ug of a2M for the data points in (a) was applied. A gradient gel(6-20% polyacrylamide) was used. Apparent Mr values (x 10-3) are indicated. A very similar pattern was obtainedafter 114Clmethylamine treatment of the samples, whereas after treatment with 1251I-labelled trypsin all samples ranas the Mr-85 000 component.

to parallel incubation mixtures, also containing['4Clmethylamine, trypsin was added at the sametime points. Incorporation of [l4C]methylamineagain showed biphasic reaction kinetics (Fig. 2).Addition of trypsin was accompanied by an in-creased [14Cimethylamine incorporation into a2M,but this was only significant in the first, rapid, phaseof incorporation. The slow-phase component, pre-viously shown to parallel the conformational changefrom electrophoretic slow-form into fast-form a2M(Van Leuven et al., 1981a), was not appreciablyaffected by formation of trypsin complex. When thepreparations obtained after 2 h incubation with[14Clmethylamine were kept under the same condi-tions for 18h, it was found that P14Clmethylamineincorporation had increased from 350 to 624c.p.m./,ug of a2M, as expected (Fig. 2). If the sample wastreated with trypsin, incorporation of F14Clmethyl-amine remained at the 2h value (362c.p.m./,ug). Bysodium dodecyl sulphate/polyacrylamide-gel electro-phoresis after denaturation and reduction, it was

found that, in all samples treated with trypsin, a2Mwas present as the Mr-85000 component (resultsnot shown). These results indicate that, althoughformation of trypsin complex stimulates incorpora-tion of ['4C lmethylamine, once the complex isformed no further incorporation occurs.

Effect ofmethylamine on trypsin bindingIn view of the foregoing and our previously de-

scribed findings (Van Leuven et al., 1981a,b), wewanted to examine further the inactivation of, a2Mby methylamine as a function of time, with respectto trypsin binding and conformational change ofa2M. Change in conformation, indexed by the shiftin electrophoretic mobility from slow-form to fast-form a2M on 5% polyacrylamide gels, was studiedwith '251I-labelled a2M. Incubation with methylamine(25mM) decreased the amount of a2M (1251 radio-activity) in slow form with time (Fig. 3). The radio-activity was quantitatively found in fast-form a2M,inasmuch that at each data point over the entire

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Primary amines and a2-macroglobulin

c4

o03

9) I,x22

a- b

0 1 2 3 4

Time (h)Fig. 2. Time course of incorporation of "4Clmethylamine

in a2M and effect offormation ofcomplex with trypsinIncubation of a2M (2mg/ml) with 25 mM-['4Cl-methylamine was in 5OmM-Tris/HCl buffer, pH 8.0,at 25 0C. At the indicated time points, samples wereeither directly applied to 5% polyacrylamide gelsor treated with trypsin (molar ratio to a2M, 3 :1),by addition of a concentrated solution of the enzyme(20 mg/ml) so that dilution of methylamine wasnegligible. After 10min samples were applied to 5%polyacrylamide gels. After electrophoresis, radio-activity in a2M was determined by excision of Coo-massie-Blue-stained bands, dissolution in concen-trated H202 and liquid-scintillation counting ofradioactivity. E, Incorporation of ['4Clmethylamineinto native a2M; 0, incorporation of ['4Clmethyl-amine into a2M by complex-formation with trypsinat the time points indicated. For the shortest incuba-tion time (2 min) results very similar to those de-picted were obtained by adding a2M last, to other-wise complete reaction mixtures, including trypsinor not (also see the text for details).

->80 °

=60_ *_ _0

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.0~~~~~~~o10 20 0_

N ~~~~~~~~~-__

0 1 2 3 4 5 6

Time (h)

Fig. 3. Time course ofconformational change and trypsinbinding ofa2M by methylamine

'251-labelled a2M (1 ug/ml) was incubated with25 mM-methylamine in 5OmM-Tris/HCl buffer,pH8.0, at 250C. At the indicated time points,samples were taken and analysed by rate electro-phoresis in 5% polyacrylamide gels. Coomassie-Blue-stained bands of slow-form and fast-form a2Mwere excised and radioactivity was determined(0, slow-form a2M). In a parallel experiment nativea2M was incubated with methylamine under exactlythe same conditions. At the indicated time pointssamples were taken and treated with excess of125I-labelled trypsin. Determination of radioactivitybound to a2M was determined as described aboveand represents total binding of trypsin to a2M (El).On the same samples enzymic activity was deter-mined after addition of soya-bean trypsin inhibitor(0). Results are normalized to measurements withnative a2M, not treated with methylamine, incubatedunder otherwise identical conditions.

incubation period total radioactivity recovered fromthe gel was more than 92% of the amount applied.Kinetically, the time course of conformationalchange did not follow first-order kinetics withrespect to time.A similar experiment was done with unlabelled

a2M but under otherwise identical conditions, andat each time point excess of 251I-labelled trypsin was

added. By this treatment all samples were convertedinto fast-form a2M. These preparations were thenexamined for the amount of trypsin bound, bothby determination of radioactivity present in a2Mand by determination of trypsin activity not inhibitedby soya-bean trypsin inhibitor. Radioactivity boundto a2M decreased with the duration of incubation with

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methylamine, closely paralleling the conformationalchange (Fig. 3). However, trypsin-inhibitor-resistanttrypsin activity decreased much more rapidly thanwas expected from the data on conformationalchange. After 6 h of methylamine incorporation,trypsin-inhibitor-resistant binding was decreased toless than 10% relative to that with native a2M incub-ated under identical conditions without methylamine.Kinetically, this time course was found to be first-order with respect to time. Taken together, these andprevious results indicated methylamine incorpora-tion, conformational change and trypsin binding tobe different phenomena, related kinetically in acomplicated fashion. Moreover, the difference be-tween total binding of trypsin (1251 radioactivity)and trypsin-inhibitor-resistant binding showed that

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F. Van Leuven, J.-J. Cassiman and H. Van den Berghe

Table 1. Enzvmic activitv of trvpsin and ofa2M-trvpsin complexesEnzymic activity towards [3Hlacetylated haemoglobin and benzoylarginine p-nitroanilide was measured as detailedin the Experimental section. Complexes were prepared and isolated as described in the text. In the assay withhaemoglobin activity is expressed as radioactivity (c.p.m.) not precipitated by trichloroacetic acid as liberated byl,ug of trypsin or by the equivalent of 200,ug of a2M as trypsin complexes. In the assay with benzoylargininep-nitroanilide activity is expressed as increase in A410/min with lO,ug of trypsin or the equivalent of 125,ug of a2Mas trypsin complexes. Benzamidine was used at a final concentration of 25 mM; 20,ug of soya-bean trypsin inhibitor(STI) was used per assay where indicated. Results are the means of duplicate determinations. Results from theassay with haemoglobin are corrected for a blank value of 1205c.p.m. (no trypsin or a2M added, but otherwiseidentical conditions).

a2M nativeTrypsin

+ STI+benzamidine

a2M-trypsin+ STI+benzamidine

a2M-trypsin (methylamine)+ STI+benzamidine

Assay with[3H lacetylatedhaemoglobin

(c.p.m.)31

10409108214

26361257673

83081243777

Assay withbenzoylargininep-nitroanilide(AA410/min)

0.00 10.0400.00 10.0020.0290.0300.0020.0180.0090.00 1

complexes thus formed are anomalous: some of the'251-labelled trypsin is inhibited by soya-bean trypsininhibitor. Examination of the observation revealedthat soya-bean trypsin inhibitor did not displacelabelled trypsin from a2M in these preparations, noteven with prolonged incubation with the inhibitor(up to 6 h).

In the spectrophotometric assay for trypsin en-zymic activity it was found that when these prepara-tions were examined immediately after additionof soya-bean trypsin inhibitor, or when the trypsininhibitor was added to the assay mixture in thecuvette, curvilinear tracings were obtained, whichbecame linear only after about 30 min. This is insharp contrast with either free trypsin or classicala2M-trypsin complexes, for which linear timecurves were always obtained, even when soya-beantrypsin inhibitor was added to the complete reactionmixture in the cuvette. With free enzyme, completeinhibition by soya-bean trypsin inhibitor was im-mediate on addition of the inhibitor under theseconditions. Apparently the reaction with soya-beantrypsin inhibitor in these anomalous a2M-trypsincomplexes is slowed down considerably, indicatingthat trypsin was bound or entrapped by a2M in asterically hindered fashion, but still reactive towardssoya-bean trypsin inhibitor.

These observations were taken into account, asthe data on enzymic activity (Fig. 3) all pertain tothe linear part of the reaction time course, whereassubsequent experiments (see below) were done afterreaction with soya-bean trypsin inhibitor for atleast 30min.

Characterization of trypsin-inhibitor-inhibited com-plexes

The difference between total binding of trypsin(l25l radioactivity) and trypsin-inhibitor-resistantbinding (enzymic activity) after methylamine treat-ment indicated formation of 'abnormal' complexes,in which trypsin could still be inhibited by soya-beantrypsin inhibitor. These complexes were furtherexamined for proteolytic activity towards a high-molecular-weight proteinaceous substrate and forbinding of soya-bean trypsin inhibitor to the activesite of trypsin. This was done with [3Hlacetylatedhaemoglobin and 125I-labelled soya-bean trypsininhibitor. For these experiments a2M was treatedfor 3h with methylamine under conditions used forthe kinetic studies described above. Control prep-arations were similarly treated, with omission ofmethylamine. At the end of the incubation, trypsinwas added in excess to a2M (molar ratio 4: 1). Thecomplexes were separated from excess of trypsinand methylamine by gel filtration on an UltrogelAcA-34 (LKB) column (1 cm x 60 cm). Fractionscontaining a2M were pooled and concentrated.

Proteolytic activity towards [l3Hacetylatedhaemoglobin was measured. A typical experimentis shown in Table 1, in which proteolytic activitytowards [3H]acetylated haemoglobin and amidaseactivity towards benzoylarginine p-nitroanilide arecompared for different preparations and treatments.

Neither assay detected enzyme activity in nativeisolated a2M. Control a2M-trypsin complexesshowed the typical behaviour when assayed with

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2

0

E (b) .-,-8

C.6

o 1o 20 30 40 50Vol. of effluent (ml)

Fig. 4. Binding of '25l-labelled soya-bean trypsin inhibitorto a2M-trvpsin complexes

a2M-trypsin complexes were formed either withnative a2M or after treatment of a2M with methyl-amine (see the text for experimental details). Thesecomplexes were further incubated for 60min withexcess of 125I-labelled soya-bean trypsin inhibitorand then analysed by gel filtration (Ultrogel AcA-34;column cm x 60cm; in 0.1 M-Tris/HCl buffer,pH 8.0); the flow rate was 6 ml/h, and 1 ml fractionswere collected. In (b) distribution of radioactivityis shown as obtained with trypsin complexes ofmethylamine-treated a2M (l). Fractions denotedby the horizontal bar contained a2M as detected by'rocket' immunoelectrophoresis. These fractionswere pooled, concentrated and rechromatographedon a similar column (0). In (a) distribution of radio-activity is shown under similar conditions of thesample complexes but treated with 7-amino-i-chloro-3-tosylamidoheptan-2-one (2 mM) beforeaddition of 125I-labelled soya-bean trypsin inhibitor.A pattern very similar to this was obtained withcontrol a2M-trypsin complexes (a2M not treatedwith methylamine or with 7-amino- l-chloro-3-tosylamidoheptan-2-one). Equal amounts of a2M(2 mg) and radioactivity were loaded on the columns.

benzoylarginine p-nitroanilide: no inhibition ofactivity with soya-bean trypsin inhibitor and nearlycomplete inhibition with benzamidine, a low-mole-cular-weight inhibitor. With [3Hlacetylated haemo-globin low enzymic activity was detected, inhibitedpartially by soya-bean trypsin inhibitor and nearlycompletely by benzamidine. Inhibition comparablewith that obtained with benzamidine was found withinhibitors Trasylol and 7-amino- 1-chloro-3-tosyl-amidoheptan-2-one ('TLCK') (results not shown).The complexes generated and isolated after methyl-

amine treatment I a2M-trypsin (methylamine);Table 11 showed a much higher activity towardshaemoglobin than did the control preparation,despite the fact that less trypsin was present asjudged from the measurements of activity towardsbenzoylarginine p-nitroanilide. In the latter, soya-bean trypsin inhibitor inhibited 50% of the activitypresent, and benzamidine inhibited completely.Comparable activities were found for the controland the methylamine-treated complexes whenassayed with [ 3Hlacetylated haemoglobin in thepresence of soya-bean trypsin inhibitor or benz-amidine (Table 1). These results indicate someproteolytic activity to be associated with a2M-trypsin complexes, and that much more activity wasfound after methylamine treatment.

Binding of soya-bean trypsin inhibitor to a2M-trypsin complexes was examined with 1251I-labelledtrypsin inhibitor, added to complexes prepared aftermethylamine treatment as described above. Separa-tion of a2M complexes from excess of labelled soya-bean trypsin inhibitor was done by gel filtration(Fig. 4). A peak of radioactivity was present in thevoid volume of the column, closely paralleling thedistribution of a2M, as determined by 'rocket'immunoelectrophoresis, and clearly separated fromthe bulk of radioactivity. Fractions indicated bythe horizontal bar were pooled, concentrated andre-run on a similar column. Although some redis-tribution of radioactivity was apparent (Fig. 4,broken line), more than 80% was recovered at aposition identical with that of the first separation.Chromatography in similar conditions of methyl-amine-a2M-trypsin complexes treated with 7-amino- 1-chloro-3-tosylamidoheptan-2-one beforeadditon of 251I-labelled soya-bean trypsin inhibitorand of control a2M-trypsin complexesi both resultedin patterns characterized by a much decreasedamount of radioactivity recovered in the a2M peak(Fig. 4). By polyacrylamide-gel electrophoresisunder native conditions of similar preparations,radioactivity was found associated with a2M bands,both on 5% gels and on gradient gels (6-20%).However, radioactivity was also found in positionssmeared between that of free 1251-labelled soya-beantrypsin inhibitor and a2M. This indicates that duringelectrophoresis dissociation of soya-bean trypsininhibitor occurred.As a final aspect of these anomalous complexes,

we examined the extent of covalent binding of 1251Ilabelled trypsin to a2M under control conditionsand after pretreatment with methylamine for 3 h as

described above. Covalent binding of the proteinaseto a2M, extensively reported elsewhere (Van Leuvenet al., 1981b), was determined as denaturation- andreduction-resistant labelled material, present as high-molecular-weight protein on polyacrylamide-gelelectrophoresis. Radioautography indicated that

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[ (a) 1

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complexes between 251I-labelled trypsin and a2Mpretreated with methylamine (25 mM) for 3 h did notcontain appreciable amounts of covalently boundtrypsin. Determination of radioactivity in excisedbands of the gel showed that about 10% of thetotal 1251I-labelled trypsin bound to a2M was in acovalent linkage as opposed to classical a2M-trypsincomplexes, in which 49% of the label was presentas protein of apparent Mr higher than that of the freeenzyme. Although in the procedure used in thepresent work methylamine remained present duringformation of the complex, we have shown that at thisconcentration (25 mM) inhibition of covalent bindingby the primary amine is far from complete (about33%) (Van Leuven et al., 1981b). This indicatesthat absence of covalent binding was typical forthese anomalous complexes. Moreover, this corre-lates with our finding that no extra methylamine isincorporated by formation of complex in the slowphase of the methylamine reaction (Fig. 2). Theabsence of methylamine incorporation and ofcovalent binding to the 'methylamine-reactive' siteunder these conditions indicates that at least a thirdpossible reaction at this site must be envisaged. Al-though simple hydrolysis is a likely candidate, wecannot exclude the possibility that internal reactionsin the a2M structure occur.

DiscussionThe data on kinetics of interaction of trypsin and

methylamine with human a2M, presented in thispaper, have to be considered in relation to the sub-unit structure of a2M. From the available literaturedata (Harpel, 1977; Barrett et al., 1979; Swenson &Howard, 1979; Harpel et al., 1979) and from thepresent results, human a2M can be represented asa tetramer of identical subunits (see Scheme 1).These are covalently linked in pairs by disulphidebridges, forming half-molecules, which in turnassociate non-covalently to form the native tetramer.Each subunit contains one methylamine-reactivesite, whereas two subunits must be involved to formone trypsin-binding site. In a trypsin complex onepeptide bound per subunit is proteolytically cleavedand all methylamine-reactivity has disappeared. Inthis static picture of a2M several important mechan-istic questions remain, such as what two subunitsform one trypsin-binding site; are there inter-actions between subunits during formation of com-plex and what is the sequence of events leadingto a stable complex? Although not providing definiteanswers, the present results are considered in thisrespect.

Titration of a2M with increasing amounts oftrypsin and back-titration with [14C imethylamineor 125I-labelled trypsin showed a close parallelbetween the remaining methylamine-reactive and

trypsin-binding sites in a 2: 1 ratio. Moreover, thesesites were kept intact to the expected proportion.Therefore these results indicate that, even at lowtrypsin/a2M ratios, no unaccounted loss of trypsin-binding sites occurs. Binding of trypsin is accom-panied by a conformational change of a2M, resultingin an increased electrophoretic mobility. In theseand other experiments we have never observedtrypsin (125I-labelled) in association with slow-forma2M or binding of trypsin to a2M already in thefast form. The reaction with trypsin thus is rapidand results practically immediately in electrophoreticfast-form a2M. In conditions where the proteinase/a2M ratio is low (as usually must be the case in vivo,and in titration experiments such as that reported inthe present paper), this poses a problem for thesecond trypsin-binding site to react. Therefore weconclude from our data that reaction (and complex-formation) of trypsin at one site in a2M renders thesecond site more susceptible (kinetically) to trypsinaction than in native a2M. Teleologically this situa-tion would also seem the most efficient for a bivalentproteinase inhibitor in vivo. Probably neighbourinteractions between subunits or half-molecules inthe tetramer induce faster reaction at the secondsite once the first is occupied (Scheme 1), althoughwe cannot exclude the possibility that a fast dissocia-tion-reassociation of half-molecules occurs, pre-ferentialfy re-forming tetramers composed of sub-units in the same conformation (native or modifiedby trypsin).

The inactivation of a2M with methylamine, al-though resulting in a final conformation identicalwith that of a2M-trypsin complexes (Van Leuvenet al., 1981 a), seems kinetically completely different.We have shown here and elsewhere (Van Leuvenet al., 1981a,b) that: (a) reaction with methylamineand trypsin are mutually exclusive when the treat-ment has already resulted in conformationally stablefast-form a2M (however, see below); (b) duringformation of trypsin complex the methylamine-reactive site is transiently activated; (c) incorpora-tion of methylamine shows biphasic reaction rates,and labelled slow-form a2M can be isolated.

Although we maintain our previously reachedconclusion that the proteolytic event triggers activa-tion of the methylamine-reactive site (Van Leuvenet al., 1981b), the present results show that by-passing the proteolytic event with methylamineactually seems to make the second half-moleculeless reactive: slower incorporation of methylamineafter about 50% of the sites have reacted and noappreciable incorporation of methylamine whentrypsin is allowed to react in the slow phase ofmethylamine incorporation. Apparently, methyl-amine reacts rapidly with only two out of the foursubunits in the tetramer. As this results in a com-pletely different reactivity of the other subunits (in

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Primary amines and a2-macroglobulin

EbJ-LSJ

Trypsin Trypsin

(seconds)

MA

(hours) E l(minutes)

Trypsin

(seconds)

Scheme 1. Schematic representation ofthe different reactions of trypsin and methvlamine with a2MThis scheme is an attempt to visualize our findings in this and previous papers (Van Leuven et al., 1981 a,b).The representation of a2M as a tetramer is based on literature data (Barrett et al., 1979; Swenson & Howard, 1979;Harpel et al., 1979; and references cited therein). Monomers of apparent Mr 185000 are represented by the largesquares. These are held together by disulphide bridges to form half-molecules, which combine in turn non-covalentlyto form the tetramer. As it is unknown what two subunits make up one trypsin-binding site, the connections drawnbetween monomers can either be covalent (disulphide) or non-covalent. For the model, this does not make any

difference. The 'methylamine-reactive' sites are represented by small open squares. Reaction (1) represents formationof complex with trypsin (T): the two available sites are filled up in rapid succession. The intermediate form (betweensquare brackets) is not observed. Inactivation of methylamine-reactive sites is denoted by filled squares. Arrowsbetween monomers in this form represents the postulated interactions between subunits (see the text). Broken linesdenote proteolytic cleavage of the monomers. Reaction (2) represents the inactivation of a2M by methylamine (MA).The intermediate form is still slow-form a2M, which has reacted with methylamine and can be isolated. Probablythis form, when treated with trypsin (3), leads to formation of anomalous complexes. These have proteolyticactivity towards haemoglobin, and trypsin can bind soya-bean trypsin inhibitor to its active site. The open circlesdenote the third possible reaction at the methylamine-reactive site: although these sites were inactivated, they donot bind trypsin or methylamine. Some of the sites in a trypsin complex (1) also must react in this fashion, as covalentbinding is never complete, even in the absence of methylamine. Approximate reaction rates are given (time tocomplete reaction). The final forms of a2M, obtained by the different treatments, are extensively characterized anddiscussed in the text. The thick lines between monomers in these complexes represent the increased non-covalentinteractions, as pointed out by Barrett et al. (1979).

contrast with trypsin), our data indicate that methyl-amine inactivates two subunits (in different half-molecules) that do not make up a trypsin-bindingsite (Scheme 1). The symmetry in a2M and the ex-cess of methylamine used (as opposed to titrationwith sub-saturating amounts of trypsin) are in favourof such a mechanism.

Further interpretation of the present results isdifficult without becoming highly speculative aboutthe nature of the mechanism and the sequence ofevents leading to an a2M-proteinase complex. Ourresults do indicate, however, that this sequence mustbe strictly adhered to, because reversing the normalsequence (proteolytic event followed by methyl-amine-reactive-site reaction) leads to 'abnormal'

complexes. These complexes exhibit proteolyticactivity, and bind soya-bean trypsin inhibitor tightly(although slowly) to the active site of trypsin (asshown by inhibition by 7-amino-1-chloro-3-tosyl-amidoheptan-2-one). These are sufficient criteria toestablish the presence of-'trypsin-inhibitor-inhibited'a2M-trypsin complexes. Further experiments tocharacterize their nature have so far been unsuccess-ful; these complexes did not bind to immobilizedsoya-bean trypsin inhibitor, probably owing to stericfactors. In isoelectric focusing, their pattern wasdifferent from that of classical a2M-trypsin com-plexes, but overlays with agarose containing caseindid not enable us to assign proteolytic activity tospecific bands, owing to loss of resolution in the

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128 F. Van Leuven, J.-J. Cassiman and H. Van den Berghe

agarose gels. Experiments on receptor-mediatedendocytosis by fibroblasts in culture are hamperedby lack of specific labelling of the 'trypsin-inhibitor-inhibited' complexes.

Further experimental work is needed to examinethese aspects and the mechanism of formation ofa2M complexes. Eventually these will allow us todetermine the biological significance, if any, of thepresent observations, under normal or pathologicalconditions in vivo.

This will certainly be facilitated by our findingthat a neo-antigenic site on a2M complexes is closelyrelated to the receptor-recognition site on these com-plexes. Evidence for this was obtained by use ofmonoclonal antibodies (Marynen et al., 1981).

While this work was in progress, several reportsappeared tentatively identifying the 'methylamine-reactive site' in a2M as a thioester, formed betweena y-glutamyl residue and a cysteine group (Tack etal., 1980; Sottrup-Jensen et al., 1980; Howard,1981). Although this does not allow us to interpretour findings further, it might offer alternative experi-mental approaches to examine the mechanism andthe significance of the findings described in thepresent paper.

This work was supported by a Grant from the BelgianCancer Fund (Algemene Spaar en Lijfrente Kas), byGrant 3.0043.79 (Fonds voor Geneeskundig Weten-schappelijk Onderzoek) and by Research Fund OT/VII/30 (University of Leuven). F. V. L. is a Post DoctoralResearch Fellow of the American Cystic FibrosisFoundation. The expert technical assistance of Mrs. M.Caems and Mrs. L. Stas is gratefully acknowledged.

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1982