Biochem. J. (1984) 221, 445-452 445 Printed in Great Britain

Human plasma a- proteinase inhibitor Purification by affinity chromatography, characterization and isolation of an active fragment

Anne D. GOUNARIS,* Molly A. BROWN and Alan J. BARRETT Department ofBiochemistry, Strangeways Laboratory, Worts Causeway, Cambridge CBJ 4RN, U.K.

(Received 16 January 1984/Accepted 5 April 1984)

Human plasma a-cysteine proteinase inhibitor (aCPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl--Sepharose, and high- resolution anion-exchange chromatography. The was obtained as a form of Mr about 64000 and material of higher Mr (about 100000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of M, 64000. An antiserum was raised against aCPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9mg/dl. Both low-Mr and high-Mr forms of aCPI were confirmed to be sialoglyco- by the decrease in electrophoretic mobility after treatment with neuramini- dase. aCPI was shown immunologically to be distinct from antithrombin III and a,-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. aCPI was also distinct from A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of aCPI with papain were detectable in immunoelectrophoresis, but dissociated to free and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. aCPI was found to be a tight-binding inhibitor of papain and human H and L (Ki 34pM, 1.1 nM and 62pM respectively). By contrast, inhibition of B was much weaker, Ki being about 35 uM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of aCPI with gave rise to an inhibitory fragment of Mr about 22000, which was isolated.

The best known ofthe cysteine proteinases (once known cysteine proteinases fall into the super- called '' proteinases) is papain, and most ofthe family of proteins homologous with papain (Bar- rett et al., 1984). Since this group contains the major lysosomal proteinases cathepsins B, H and Abbreviations used: the abbreviations used for names L, as well as , which is used medic- of derivatives and N-terminal groups are based on the standard conventions [Biochem. J. (1984) ally, the physiological inhibitors ofthe are 219, 345-373]. The C-terminal groups are: CH2CL, of interest. Of the small number of proteins known chloromethane, NPhNO2, p-nitroanilide, and NMec, to act as tight-binding inhibitors of these cysteine 7-(4-methyl)coumarylamide. Other abbreviations used proteinases, all are small proteins, Mr about 13000, are: Cm-, S-carboxymethyl; aCPI, plasma a-cysteine except the plasma protein, aCPI. proteinase inhibitor; E-64, L-3-carboxy-2,3-epoxypro- Human aCPI was first described by Sasaki et pyl-leucylamido-(4-guanidino)butane; f.p.l.c., fast pro- al. (1977), and further work by Ryley (1979), tein liquid chromatography (Pharmacia system); [I]t, Sasaki's group (Sasaki et al., 1981 ; Taniguchi et al., total inhibitor concentration; Ki, dissociation constant 1981) and Jarvinen (1979) showed that the for inhibitor; Ki(app), apparent dissociation constant for protein inhibitor uncorrected for competition by substrate; [S], occurs in relatively low-Mr and high-Mr forms in substrate concentration; SDS, sodium dodecyl sulphate; plasma, with C2 and al electrophoretic mobilities v0, rate; vi, rate in the presence of inhibitor. respectively. Jarvinen (1979) described the purifi- * Present address: Department of Chemistry, Vassar cation ofthe two forms by use of affinity chromato- College, Poughkeepsie, NY 12601, U.S.A. graphy on a form of immobilized papain. Vol. 221 446 A. D. Gounaris, M. A. Brown and A. J. Barrett

Experimental NMec as substrate at pH 6.0, and was Materials assayed with Arg-NMec as substrate at pH6.5 Plasma was from blood supplied by a local (Barrett & Kirschke, 1981; Barrett et al., 1982). blood-transfusion centre, and contained acid Dipeptidyl peptidase I was assayed with Gly-Phe- citrate/dextrose anticoagulant as described pre- NMec (10 gM) in 0.1OM-sodium phosphate buffer, viously (Barrett et al., 1979). Volumes of plasma pH 6.5, containing 1 mM-EDTA, 50mM-NaCl and given in the text have been corrected for dilution 1 mM-dithiothreitol. was assayed with Bz- by the anticoagulant. DL-Arg-NPhNO2 as substrate, thrombin with Cm-papain-Sepharose (0.6mg of papain/g wet Boc-Val-Pro-Arg-NMec, and plasma kallikrein wt. ofgel) was prepared as described by Anastasi et with Z-Phe-Arg-NMec, all as described by Nagase al. (1983), and Cm-chymopapain-Sepharose was & Barrett (1981). prepared in an analogous manner. Chymopapain Quantification of inhibitory activity was partially purified from latex of Carica by a salt fractionation procedure based on that of The procedure used for titration of aCPI with Brocklehurst et al. (1981). Papain was isolated papain was one previously described for , from Carica papaya latex by a modification of the in which the substrate was Z-Phe-Arg-NMec method of Baines & Brocklehurst (1979). Ficin (Anastasi et al., 1983). (twice-crystallized) was from Sigma (London) Ki values were determined by use of continuous Chemical Co. Bromelain (stem) was the crystalline fluorimetric assays in a Perkin-Elmer LS-3 suspension from Boehringer Corp. (London) Ltd. spectrofluorimeter standardized with 0.2 MM- Human cathepsins B and H were isolated as aminomethylcoumarin for readings at A, 360nm described by Schwartz & Barrett (1980). Human and Aexc. 460nm. The substrates (as used in assays: was purified from a homogenate of see above) were at 10Mm final concentration, and human liver by acid treatment, acetone fractiona- all measurements were made with less than 2% tion, gel chromatography and f.p.l.c. ion-exchange hydrolysis. For and cathepsin H, Km chromatography (G. D. J. Green & A. J. Barrett, was known to be much greater than 10,UM, with the unpublished work). Human plasma kallikrein respective substrates (Barrett & Kirschke, 1981). and antiserum against it (which also reacts with For human cathepsin L, Km for Z-Phe-Arg-NMec prokallikrein) were available in the laboratory was found to be 2 pM by the method of Wilkinson (Nagase & Barrett, 1981), as were the human liver (1961) (A. J. Barrett, unpublished work). For cysteine-proteinase inhibitors and papain and dipeptidyl peptidase I, v0/[S] was , and antisera against them (Green et al., constant in the range 5-20 M substrate, so it was 1984). Bovine spleen dipeptidyl peptidase I, kindly concluded that Km > 10Mm. The concentrations of given by Dr. J. K. McDonald (Medical University the enzymes during the assay were approx. 0.02nM of South Carolina, Charleston, SC, U.S.A.), was (cathepsin B), 0.1 nm (human cathepsin L), 0.1 nM further purified by f.p.l.c. Neuraminidase (Vibrio (cathepsin H), 0.01 nM (papain) and <0.1 nM cholerae; 500 units/ml) was from Koch-Light (dipeptidyl peptidase I). Laboratories. Bovine trypsin (type XII) and bovine The method was as follows. The fluorimeter thrombin were from Sigma. cuvette and all solutions were pre-warmed to 30°C Samples of purified aCPI (mixed high-Mr and (20°C for cathepsin L). Into the cuvette was placed low-Mr forms) and anti-(aCPI) serum for compari- 40ul of stock enzyme solution and 40M1 of 100mM- sons were kindly given by Dr. H. C. Ryley (Welsh dithiothreitol. A 1-2 min period was allowed for National School of Medicine, Cardiff, U.K.). activation, and then 3.84ml of the appropriate Antiserum to papain was kindly given by Dr. E. assay buffer and 40pl of 1 mm substrate solution in Shapira, The Children's Memorial Hospital, Chi- dimethyl sulphoxide were thoroughly mixed in. cago, IL, U.S.A. Antisera against antithrombin Once a stable reaction rate had been recorded, the III, al-antichymotrypsin and haptoglobin were inhibitor was added in 40Ml and the whole mixed purchased from Behringwerke A.G. The non-ionic well. The reaction rate was followed for up to detergent Brij 35 was purchased from BDH 60min as it relaxed progressively to the new linear Chemicals, and Ultrogel AcA-44 from LKB rate corresponding to the concentration of enzyme Instruments. Di-isopropyl phosphorofluoridate in equilibrium with the enzyme-inhibitor com- was from Sigma, and Pro-Phe-Arg-CH2CI was a plex. Control experiments were made to detect gift from Dr. E. N. Shaw, Brookhaven National spontaneous decay of enzymic activity during the Laboratory, Upton, NY, U.S.A. period of the experiment. Typically, data were obtained for five inhibitor concentrations. Ki Enzyme assays values were determined from re-plots of the form Papain, bromelain, cathepsin B and cathepsin L [I],/(1-vi/vo) versus vo/vi (Henderson, 1972). When were assayed fluorimetrically with Z-Phe-Arg- experiments were made with [S] < Kn, corrections 1984 a-Cysteine proteinase inhibitor 447 were made by use of the relationship Ki ing 0.5M-NaCl and 0.1% Brij 35. The column was = Ki(app.)/(1 + [S]I/K) for simple competition. washed with 500ml of the citrate buffer followed by about 2 litres of 0.05M-potassium phosphate Raising of antiserum buffer, pH8.5, also containing the NaCl and Brij A sheep was injected on three occasions at 2- 35, to lower the A280 of the effluent to less than week intervals; on each occasion, 1 mg of purified 0.05. The inhibitor was eluted with 0.05M-potas- low-Mr aCPI in 1 ml was emulsified with an equal sium phosphate/NaOH, pH 11.5, also containing volume of Freund's adjuvant, and injected intra- the NaCl and Brij 35. Effluent fractions (5 ml) were muscularly in the thigh. On the first occasion, collected into tubes containing 1 ml of 0.25M- complete adjuvant was used, and subsequently KH2PO4 to effect rapid neutralization of the incomplete adjuvant. The animal was bled 2 weeks solution. Fractions containing the peak of protein after the third . (A280) eluted with alkali were combined, concen- trated to 10ml by ultrafiltration, and transferred Immunoelectrophoresis, immunodiffusion and 'rocket' into 50mM-Tris/25mM-HCl buffer, pH 8.0. electroimmunoassay On some occasions, a smaller (60cm3) column of These techniques were performed as described Cm-chymopapain-Sepharose was used in the same by Barrett (1974) and Barrett et al. (1979). The way, but with smaller volumes of the wash 'rocket' immunoassay was standardized with low- solutions. Mr aCPI, assuming AI'0o = 5.9 (Ryley, 1979), and Anion-exchange chromatography. The material it was also assumed that all forms of aCPI reacted eluted from the Cm-papain-Sepharose column at equally in the assay. pH 11.5 comprised the low-Mr form of aCPI, high- Mr forms ofaCPI, and minor contaminants. These Polyacrylamide-gel electrophoresis were separated by anion-exchange chromato- SDS/polyacrylamide-gel electrophoresis was graphy. The Mono Q anion-exchange column done by the method of Bury (1981) in gels of 7% or (5cm long) was used in a Pharmacia f.p.l.c. system. 12.5% polyacrylamide. Samples were either re- The linear gradient increased from 0.05M-Tris/ duced with 2-mercaptoethanol, or run unreduced 0.025M-HCI to 0.50M-Tris/0.25M-HCl, pH about after treatment with iodoacetate, as described by 8.0, at 22°C, after the sample had been applied in Barrett et al. (1979). Mr calibration was with the starting buffer. Some protein passed unad- phosphorylase b (M, 94000), transferrin (Mr sorbed through the column, and a second minor 78000), bovine serum albumin (Mr 68000) peak (peak I) was eluted at 0.1OM-Cl-, followed by immunoglobulin G heavy chain (M, 50000), three complex major peaks (II, III and IV) (Fig. 1). carbonic anhydrase (M, 29000), immunoglobulin Unadsorbed material, and that in peak I, lacked G light chain (Mr 25000), soya-bean trypsin inhibitory activity, but contained an enzyme that inhibitor (Mr 21000), cytochrome (Mr 12750) and hydrolysed Z-Phe-Arg-NMec at neutral pH; this aprotinin (M, 6500). Mr values for unknown was inhibited by di-isopropyl phosphorofluoridate components were calculated by use of linear plots and Pro-Phe-Arg-CH2Cl, and was identified as of logM, versus migration distance. plasma kallikrein by double immunodiffusion Pore-limit gradient gel electrophoresis of native against anti-kallikrein serum in comparison with proteins was done at pH9.1 in gels of 4-26% poly- an authentic sample. We have no explanation for acrylamide as described by Barrett et al. (1979). the affinity of kallikrein (or perhaps prokallikrein) for Cm-papain-Sepharose. Ultrafiltration Inhibitory activity was detected in effluent This was done in an Amicon cell fitted with a fractions comprising peaks II and IV, but not peak PM-10 or PM-30 membrane, under pressure of N2. III. Samples from fractions 32 (peak II) and 46 (peak IV) were run in SDS/polyacrylamide-gel electrophoresis (Fig. 2; see below), and the two peaks of inhibitory activity were found to cor- Results respond to the low-Mr and high-Mr forms respec- tively of aCPI. In a typical purification we Purification of aCPI obtained 27.8mg of low-Mr aCPI and 13.5mg of aCPI was purified from plasma in two steps, high-Mr oCPI from 400ml of plasma. affinity chromatography on Cm-papain-Sephar- ose and anion-exchange chromatography. Immunochemical tests Affinity chromatography. Plasma (400ml) was Tests were made to establish the concentration run at 300ml/h on to a column (l2cmx4.5cm, of aCPI in serum, to discover whether any other 190cm3) of Cm-papain-Sepharose equilibrated plasma proteins that might be inhibitors of with 0.05M-sodium citrate buffer, pH 6.5, contain- cysteine proteinases have affinity for Cm-papain- Vol. 221 448 A. D. Gounaris, M. A. Brown and A. J. Barrett

II I'

_ 0.4 0.2 -'

._ S. CZ 0 IV 04: too

III 0.1 U

40 Fraction no. Fig. 1. Mono Q fp.l.c. of aCPI The inhibitory material (5ml) from the Cm-papain-Sepharose affinity column was run on the Mono Q column equilibrated with 0.05 M-Tris/0.025M-HCl buffer, pH 8.0, and eluted with a linear gradient to 0.50M-Tris/0.25M-HCl buffer, pH 8.0, at 22°C. The bars indicate points at which samples were taken for SDS/polyacrylamide-gel electro- phoresis (Fig. 2). , A280; --, Cl- gradient.

Sepharose, and whether the purified aCPI frac- The absence ofreactivity for antithrombin III in tions have immunological reactivities of any other the aCPI samples shows that aCPI is quite distinct proteins. from the thrombin inhibitor, although the latter Concentration of ctCPI in human serum. Nineteen has been reported to be an inhibitor of papain samples of serum from hospital patients were run (Valeri et al., 1980). In view of the fact that it is in 'rocket' immunoelectrophoresis in agarose gel very unusual for a protein inhibitor to be active containing anti-aCPI serum, together with stan- against proteinases ofmore than one catalytic class dard samples of aCPI. It was found that the con- (Laskowski & Kato, 1980), it may be necessary to centration was 35.9mg/dl (range 24.0-48.8mg/dl). consider whether the inhibition of papain by some Test of aCPI preparations for other proteins. A preparations of antithrombin is due to contamina- solution of crude aCPI from the Cm-papain- tion with aCPI. aCPI also was clearly distinct from Sepharose column (1 mg/ml), plasma that had a1-antichymotrypsin. The immunological dissimi- passed through the column, and purified aCPI larity of oaCPI from the low-Mr inhibitors from (1 mg/ml) were run in double immunodiffusion liver was further confirmed by the finding that against antisera to haptoglobin, antithrombin III, antiserum to aCPI did not react with the human human liver cystatins A and B and total human cystatins in double immunodiffusion. serum proteins. Of these antigens, only aCPI and plasma kallikrein were detected in the fraction Characteristics of the molecular forms of aCPI eluted from the affinity column, and only aCPI in Experiments were made in an attempt to clarify the purified preparation. The plasma that had the structural relationship between the low-Mr passed through the affinity column was conspicu- form of aCPI and the two forms that have greater ously depleted of aCPI and kallikrein (or pro- kallikrein), but not of haptoglobin, antithrombin Mr and more negative molecular charge. III or ac-antichymotrypsin. SDS/polyacrylamide-gel electrophoresis. Frac- Rather surprisingly, the commercial antiserum tions from the Mono Q anion-exchange column to whole human serum did not react with cxCPI. (Fig. 1) were run in SDS/polyacrylamide-gel Immunoelectrophoresis ofthe high-Mr and low-Mr electrophoresis with and without reduction (Fig. forms of aCPI showed no precipitin arcs (suggest- 2). Without reduction, material from pool II ran ing the absence of gross contamination by other with Mr 64000, whereas pool IV showed two serum proteins), whereas plates developed with the components of Mr about 100000. With reduction, anti-aCPI serum produced arcs confirming the a2- both pools gave a major component of Mr 64000, and a I-mobilities respectively of the low-Mr and with minor components of lower Mr. These are high-Mr forms reported by Sasaki et al. (1981). formed by the degradation of aCPI by a proteinase 1984 a-Cysteine proteinase inhibitor 449

phoresis, and the plates developed with antiserum = : =~~~~~~nmm_ against ocCPI. The mobilities of both forms of the inhibitor were decreased by the action of neura-

~ -_- minidase, and we concluded that aCPI is a sialoglycoprotein, in agreement with the report by Ryley (1979). Desialylation did not cause the forms to acquire the same electrophoretic mobility, however.

Interaction of aCPI with proteinases _ Detection of the aCPI-papain complex. Papain, - Cm-papain, ocCPI (low-Mr form), and two mix- tures, one aCPI with papain, the other aCPI with (a) (b) (c) (d) (e) (a) Cm-papain, were run in immunoelectrophoresis. The plate was developed with antisera against Fig. 2. SDS/polyacrylamide-gel electrophoresis ofsamples papain and azCPI (Fig. 3a). The precipitin arcs for from Mono Q fp.l.c. papain and aCPI against their respective antisera Samples were Mr markers (see the Experimental so d their reneectrophortic section) run reduced (a), fraction 32 run unreduced showed their widely different electrophoretic (b) and reduced (c), and fraction 46 run unreduced mobilities. Each mixture showed a new precipitin (d) and reduced (e). It can be seen that the major arc of intermediate mobility that was antigenic for bands in (b), (c) and (e) run between immuno- both antisera, and was thus identified as an cxCPI- globulin G heavy chain and serum albumin, with Mr papain complex or as an ocCPI-Cm-papain com- about 64000. plex respectively. The complex between aCPI and (carboxy- methylated) papain was also demonstrated in pore- limit electrophoresis (Fig. 3b). SDS/polyacrylamide-gel electrophoresis ofaCPI- associated with it in some preparations (G. S. papain complexes. aCPI (100pg) and papain Salvesen & A. J. Barrett, unpublished work). (100.g) were mixed in 100pI of 0.05m-sodium Gel chromatography. Partially purified aCPI citrate buffer, pH6.5, containing 0.5M-NaCl, 0.1°% from the Cm-papain-Sepharose column was run 35 and was then on a column (85 cm x 1.5 cm, 150cm3) of Ultrogel Brij 2mM-cysteine. Iodoacetate Ac-4 in 0.0msdu citat bufr pH.5 added in excess over the cysteine, to inactivate the containingconaiin 0.50.5M-NaCl andan 0.10.1% Brij 35. Three proteinase,polyacrylamide-gelbefore electrophoresisthe mixture waswithrunreduction,in SDS/ peaks of protein.-aCwere obtained, theBrj3..hesecond and i a 12.5%-polyacrylamideg gel.similarexperi- third of which inhibited papain, and gave a mn w madeywlth ficin and catheSsin B in precipitin reaction with anti-aCPI serum. When place of Always, the bands on the gel cor- run in SDS/polyacrylamide-gel electrophoresis papain.to those ofthe free inhibitor and the free with and without reduction, peak-2 material ran responded exactly like the high-Mr, more-retarded, material enzyme. Wereactionconcludeddoes notthatinvolvethe aCPI-cysteinethe formation from Mono whereas III was shown to cor- proteinase Q, peak of a covalent SDS-stable complex, or cleavage of respond to the low-Mr, less-retarded, fraction. the inhibitor molecule, both of which have been These results suggested that the relative Mr values seen with plasma inhibitors of serine proteinases indicated by SDS/polyacrylamide-gel electro- such as ap-proteinase inhibitor and antithrombin phoresis ofthe forms do correspond to those of the III (Travisis & Salvesen, 1983). natiuP114LIV; PLLUSllb.nrntpLineI Effect of neuraminidase on aCPI. The greater negative charge of the high-Mr forms of aCPI Stoicheiometry of the ocCPI-papain interaction relative to the lower-Mr form (seen in immuno- This was estimated in two ways: by direct electrophoresis and anion-exchange chromato- titration of aCPI with papain, and by examination graphy) is not directly attributable to the differ- of the molecular size of the complexes. ences in Mr, but might have been due to Titration of aCPI with papain. Solutions of differences in sialylation. purified low-Mr and high-Mr forms of oCPI were Samples (50pg) of the low-Mr and high-Mr titrated with papain that had itself been standard- forms of aCPI were treated with neuraminidase ized with E-64 (Barrett et al., 1982). On the basis of (10 Koch-Light units) in 0.1 ml of sodium acetate Al 96 = 5.7 (Ryley, 1979), it was calculated that the buffer, pH5.5, containing 6mM-CaCl2 for 90min preparations contained one inhibitory site per at 37°C. Samples were run in immunoelectro- 73600 and 92400 daltons of protein respectively. Vol. 221 450 A. D. Gounaris, M. A. Brown and A. J. Barrett

(a) at 25°C for 1 h before removal of samples for gel electrophoresis. Two protein bands were observed for aCPI incubated with glutaraldehyde in the absence of papain, with apparent M, values of 54700+ 1300 60|(i} (iii) (iv}\ (v) (S.E.M., n = 7) and 97000 (n = 2). These were interpreted as representing aCPI monomer in |*\ 11~~~~~~~~~~~~~~~.....~~~~~~~~~~~~~~~~~which intrachain cross-linking had produced a more compact molecule than the unfolded normal

I protein, and an interchain-cross-linked aCPI / dimer respectively. The Mr of native cxCPI was 64000 + 2700 (S.E.M., n = 3). Similarly, monomer (. ) and dimer bands were observed for papain cross- linked with glutaraldehyde. One new band was observed in all incubations of aCPI with papain or Cm-papain in the presence of glutaraldehyde. The apparent Mr of this was + 74800+2200 (S.E.M., n = 3). The sum of the M, A B A values observed for glutaraldehyde-treated papain, 24200 + 1200 (S.E.M., n = 3), and glutaraldehyde- (i.) (ii)l ii) treated ocCPI, 54700+1300 (S.E.M., n=7) was 78900. This new component was interpreted as a (b) glutaraldehyde-cross-linked complex of aCPI and papain in a 1 :1 molar ratio.

Affinity of ctCPIfor proteinases TfnTfn- _ aCPI proved to be a tight-binding inhibitor of papain, cathepsin H and cathepsin L, the respec- tive Ki values being 34pM, 1.1nM and 62pM. By contrast, the inhibition of cathepsin B was much Bsa- weaker, the slight inhibition obtained at the + highest inhibitor concentration used (13nM) being compatible with a Ki of0.35 pM. Similarly, dipepti- Fig. 3. Electrophoretic detection of aCPI-papain dyl peptidase I was weakly inhibited, with Ki complexes approx. 0.13 gM. (a) Immunoelectrophoresis. Well (i), Cm-papain Low concentrations of aCPI partially inhibited (4jpg); well (ii), Cm-papain (4pg) with aCPI (2kg); well (iii), aCPI (4 ig); well (iv), papain (3.5pg) with commercial bromelain, but very high concen- aCPI (2ug); well (v), papain (3.5pg). Troughs A trations were required to complete the inhibition. contained anti-papain serum, and troughs B con- This was found to be due to heterogeneity of the tained anti-aCPI serum. (b) Pore-limit electrophore- commercial bromelain, the major protein compo- sis. Samples run in the gradient of increasing poly- nent being a cysteine proteinase that was very acrylamide concentration (from top down) were (i) weakly inhibited by czCPI, with Ki(app.) 0.21 gM standards human transferrin (Tfn) and bovine (A. J. Barrett, unpublished-work). serum albumin (Bsa), as indicated, (ii) aCPI (low- No inhibition of the serine proteinases trypsin with excess Cm- M, form), and (iii) aCPI mixed and thrombin by aCPI was detected. The activity papain. It can be seen that all of the aCPI was of plasma kallikrein was directly detectable in converted into less-mobile complex with the enzyme derivative. crude aCPI preparations, showing that this serine proteinase also was not inhibited.

Preparation of inhibitory fragment from aCPI aCPI (50mg) partially purified by affinity Glutaraldehyde-cross-linking of aCPI-papain chromatography on Cm-chymopapain-Sepharose complexes. Low-Mr aCPI (115ug/ml), papain was incubated with 1 mg of commercial bromelain (30pg/ml), and a mixture of the two, all in 30mM- in a total volume of 5 ml of 0. I0M-sodium phos- sodium citrate buffer, pH6.5, containing 0.30M- phate buffer, pH6.5, containing 1 mM-EDTA. The NaCl and 0.03% Brij 35, were incubated with bromelain had been pre-activated in mM-dith- glutaraldehyde (10mM) for 10min at 40°C and left iothreitol, and the concentration of dithiothreitol 1984 a-Cysteine proteinase inhibitor 451

separated the high-Mr and low-Mr forms of aCPI, as well as removing contaminants. The first peak ofaCPI eluted from the Mono Q was the species of Mr 64000 that has the lesser anodal mobility in gel electrophoresis, and has been called 'x2-thiol proteinase inhibitor'. The aCPI eluted later from Mono Q corresponded to the more-anodal and higher-Mr form that has been called 'ax-thiol proteinase inhibitor'; we found this to be hetero- geneous, consisting of about two species, with M, 80000-100000, in SDS/polyacrylamide-gel electro- phoresis under non-reducing conditions. The inhibition of cathepsin B by serum was attributed largely to a2-macroglobulin in the study by Starkey & Barrett (1973), although there was apparent inhibition by a component of similar M, (a) (b)(c) (d) (e) to immunoglobulin G. This may have been the Fig. 4. SDS/polyacrylamide-gel (12.5%, reduced) electro- result of proteins acting as competing substrates in phoresis of bromelain digest of acCPI the assays. Our present results show that the Ki of Lane (a), cxCPI (a somewhat aged preparation); lane aCPI for cathepsin B is only about an order of (b), bromelain digest; lane (c), Ultrogel AcA-44 magnitude below its plasma concentration, so that chromatography peak 4; lane (d), f.p.l.c. Mono Q assays with diluted column fractions and a sub- fraction; lane (e), standards (see the Experimental strate competing for cathepsin B would not have section). shown distinct inhibition by aCPI. The physiological function of aCPI remains to be established. The molar concentrations of a2- during the digestion was 0.01 mm. The digestion macroglobulin and aCPI in plasma are approx. was for 30min at 30°C, and the solution was then 3.4 IM and 5.5 iM respectively, but aC2-macro- made 10% (w/v) with respect to sucrose, and globulin binds two molecules of papain per applied to a column (1.5cm x 84cm) of Ultrogel molecule (Howell et al., 1983). Sasaki et al. (1983) AcA-44 equilibrated with 0.1 M-sodium phosphate have suggested that inhibition of in the buffer, pH6.5. Four major peaks of protein (as blood may be mediated predominantly by aCPI. A280) were eluted, the third of which comprised This could be important in view of the marked material of Mr about 20000 that was inhibitory for effects of calpain on platelet function reported by papain. Yoshida et al. (1983). Unlike a2-macroglobulin, Fractions representing the inhibitory peak were aCPI is almost certainly ofsmall enough molecular combined, dialysed against 0.02M-Tris/HCl size to escape from the blood into the tissues, where buffer, pH 8.0, and run on the Mono Q column of it could regulate extracellular activities of the the Pharmacia f.p.l.c. apparatus in a gradient of lysosomal proteinases. the same buffer from 0.02M- to 0.50M-Tris/HCl. The A280 profile of the effluent presented a Much of this work was done while A. D. G. was Ann complex pattern of peaks, including about five Horton Research Fellow, Newnham College, Cam- major ones, the fourth of which was a doublet. The bridge, 1980-81. We thank Dr. M. E. Davies for material comprising this fourth peak was re-run on performing the test of immunological cross-reaction the Mono Q column under the same conditions; between aCPI and the liver cysteine proteinase inhibi- again the peak was double-headed, but the tors, Miss Wendy Webb for the 'rocket' immunoelectro- phoresis, Mr. Neil Rawlings for much help with the material in the later portion had the higher specific preparation of aCPI, and Miss Rosalind Hembry for inhibitory activity, and was found to consist immunization of sheep. We thank our colleagues for primarily of a single component of Mr about 20000 many stimulating discussions. when run in SDS/polyacrylamide-gel electrophore- sis (Fig. 4). References Discussion Anastasi, A., Brown, M. A., Kembhavi, A., Nicklin, M. J. H., Sayers, C. A., Sunter, D. C. & Barrett, The two-step procedure described for the purifi- A. J. (1983) Biochem. J. 211, 129-138 cation of xCPI from human plasma is simpler than Baines, B. S. & Brocklehurst, K. (1979) Biochem. J. 173, those of Sasaki et al. (1977) and Ryley (1979). 541-548 F.p.l.c. on the Mono Q anion-exchange column Barrett, A. J. (1974) Biochim. Biophys. Acta 371, 52-62 Vol. 221 452 A. D. Gounaris, M. A. Brown and A. J. Barrett

Barrett, A. J. & Kirschke, H. (1981) Methods Enzymol. Ryley, H. C. (1979) Biochem. Biophys. Res. Commun. 89, 80, 535-561 871-878 Barrett, A. J., Brown, M. A. & Sayers, C. A. (1979) Sasaki, M., Minakata, K., Yamamoto, H., Niwa, M., Biochem. J. 181, 401-418 Kato, T. & Ito, N. (1977) Biochem. Biophys. Res. Barrett, A. J., Kembhavi, A. A., Brown, A. A., Commun. 76, 917-924 Kirschke, H., Knight, C. G., Tamai, M. & Hanada, Sasaki, M., Taniguchi, K. & Minakata, K. (1981) J. K. (1982) Biochem. J. 201, 189-198 Biochem. (Tokyo) 89, 169-177 Barrett, A. J., Nicklin, M. J. H. & Rawlings, N. D. Sasaki, M., Taniguchi, K., Suzuki., K. & Imahori, K. (1984) Acta Biochim. Biophys. Acad. Sci. Hung. in the (1983) Biochem. Biophys. Res. Commun. 110, 256- press 261 Brocklehurst, K., Baines, B. S. & Kierstan, M. P. J. Schwartz, W. N. & Barrett, A. J. (1980) Biochem. J. 191, (1981) Biotechnology 5, 262-336 487-497 Bury, A. F. (1981) J. Chromatogr. 213, 491-500 Starkey, P. M. & Barrett, A. J. (1973) Biochem. J. 131, Green, G. D. J., Kembhavi, A. A., Davies, E. M. & 823-831 Barrett, A. J. (1984) Biochem. J. 218, 939-946 Taniguchi, K., Ito, J. & Sasaki, M. (1981) J. Biochem. Henderson, P. J. F. (1972) Biochem. J. 127, 321-333 (Tokyo) 89, 179-184 Howell, J. B., Beck, T., Bates, B. & Hunter, M. J. (1983) Travis, J. & Salvesen, G. S. (1983) Annu. Rev. Biochem. Arch. Biochem. Biophys. 221, 261-270 52, 655-709 Jarvinen, M. (1979) FEBS Lett. 108, 461-464 Valeri, A. M., Wilson, S. M. & Feinman, R. D. (1980) Laskowski, M., Jr. & Kato, I. (1980) Annu. Rev. Biochem. Biochim. Biophys. Acta 614, 526-533 49, 593-626 Wilkinson, G. N. (1961) Biochem. J. 80, 324-332 Nagase, H. & Barrett, A. J. (1981) Biochem. J. 193, 187- Yoshida, N., Weksler, B. & Nachman, R. (1983) J. Biol. 192 Chem. 257, 7168-7174

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