[55] Human α1-proteinase inhibitor

754

INHIBITORS OF VARIOUS SPECIFICITIES

[55]

nents, respectively) are of similar M,. to the ~2M subunit, and that the locations of their proteolysis-sensitive and covalent-linking/autolysis sites correspond closely to those in ~2M (Fig. 4). It thus seems that there is a strong possibility of evolutionary homology between ~2M, C3, C4, and possibly C5. In another approach to the evolution of c~2M, Starkey and I have surveyed the chordate phyla for proteins with the characteristic properties of c~zM, using as our primary test the capacity to bind papain in a form active against low-M, substrates? 4 The mammals, birds, reptiles, and amphibia all seem to have an ~2M-like protein of similar molecular size to human c~2M. The corresponding protein in the fish, however, has a molecular size corresponding to a half-molecule of o~2M. A study of its structure indicates that it is not simply the dimer of subunits of about 180,000 Mr, but that each precursor polypeptide chain is further cleaved just N-terminal to the proteolysis site, as it is in human C3. The protein nevertheless operates the trap mechanism, and binds methylamine covalently. ;~ P. M. Starkey and A. J. Barrett, unpublished results (1980).

[55] By

Human al-Proteinase Inhibitor JAMES T R A V I S a n d D A V I D J O H N S O N

Human plasma is known to contain at least seven proteinase inhibitors, ~'e many of which are believed to have specific functions in the coagulation, fibrinolytic, or complement pathways. However, the major proteinase inhibitor in plasma, referred to as either ~rproteinase inhibitor (aa-PI) or C~a-antitrypsin, apparently acts as a general scavenger for tissue serine proteinases. This protein was first isolated by Schultze et al.3 and referred to as the 3.5 S ~i-glycoprotein of plasma. It was found by Jacobsson to be the major trypsin inhibitor in plasma 4 and renamed %-antitrypsin in 1962.~ However, because of the broad spectrm of inhibitory activities of this protein and, in particular, its apparent major funcN. Heimburger, H. Haupt, and H. Schwick, Proc. Int. Res. Col~f~ Proteinase Inhibitor.s, 1st. 1970 p. 1 (1971). '-' M. Moroi and N. Aoki, J. Biol. Chem. 251, 5956 (1976). :~ H. E. Schultze, I. Gollner, K. Heide, M. Schonenberger, and G. Schwick, Z. Naturjbrsch.. B: Anorg. Chem.. Org. Chem.. Biochem., Biophys.. Biol. 10B, 463 (1955). K. Jacobsson, Stand. J. Clin. Lab. Invest.. Suppl. 14, 55 (1955). H. E. Schultze, K. Heide, and H. Haupt, Kiln. Wochenschr. 40, 427 (1962).

METHODS IN ENZYMOLOGY,VOL. 80

Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181980-9

[55]

HUMAN OLI-PROTEINASE INHIBITOR

755

tion to control the activity of neutrophil proteinases, it was renamed al-proteinase inhibitor. 6 Major interest in a r P I began in 1963 when Laurell and Eriksson 7 noted a deficiency in plasma levels of this protein in several patients who had developed e m p h y s e m a . These individuals synthesize an aberrant form of c~rPI that is poorly transported into the circulation. It is now known, in fact, that a l - P I can exist in at least nine forms known as Pi variants, with the Pixi protein present in most individuals with normal plasma levels of ~ - P I (130 mg a r P I / 1 0 0 ml plasma~'~). In contrast, the Piz protein is found primarily in individuals with low plasma al-PI levels (less than 20 mg/100 ml plasma). The difference between the M and Z proteins has been described and is primarily due to the substitution of a glutamic acid residue in the M protein by a lysine residue in the Z protein. 1°'~ The fact that low cq-PI levels in plasma can be correlated with emp h y s e m a development has stimulated an intensive study of the role of this protein in the maintenance of a proper p r o t e i n a s e - p r o t e i n a s e inhibitor equilibrium in tissues. In fact, the protein has now been purified by at least six different procedures, using (NH4)2804 fractionation, gel-filtration c h r o m a t o g r a p h y , ion-exchange c h r o m a t o g r a p h y , affinity chromatography, and preparative gel electrophoresis to isolate the p r o t e i n . ~'~'"'~'~2 t:~ Assay Methods Assays are based on the inhibition of proteinase activity resulting from preincubation of the e n z y m e with inhibitor. Since aa-PI inhibits a variety of serine proteinases, the assay procedures actually depend on the proteinase being inhibited. Historically, trypsin has been employed for the assay of al-PI because it is available in high purity and easily measured using synthetic substrates. H o w e v e r , a l - P I apparently functions in v i v o as an inhibitor o f p o l y m o r p h o n u c l e a r leukocyte elastase. 1~ Because this en'; R. Pannell, D. Johnson, and J. Travis, Biochemistry 13, 5439 (1974). C. B. Laurell and S. Eriksson, Stand. J. Clin. Lab. Invest. 15, 132 (1963). J. O. Jeppson, C. B. Laurell, and M. Fagerhol, E,r. J. Biochem. 83, 143 (1978). ~' M. K. Fagerhol, Set. ttaematol. 1, 153 (1968). ~"A. Yoshida, J. Lieberman, L. Gaidulis, and C. Ewing, Proc. Natl. Acad. Sci. U.S.A.

73, 1324 (1976). ~JJ. O. Jeppson, FEBS Lett. 65, 195 (1976). ~zI. P. Crawford, Arch. Biochem. Biophys. 156, 215 (1973). ~:~1. E. Liener, O. R. Garrison, and Z. Pravda, Biochem. Biophys. Re,,. Commlttt. 51, 436 (1973). ~4p. Musiani, G. Massi, and M. Piantelli, Clin. Chim. Acta 73, 561 (1976). ~ M. Plancot, A. Delacourte, K. K. Han, M. Dautrevaux, and G. Biserte, Int. J. Pept. Protein Reds. 10, 113 (1977). ~; K. Beatty, J. Bieth, and J. Travis, J. Biol. Chem. 255, 3931 (1980).

756

INHIBITORS OF VARIOUS SPECIFIC1TIES

[55]

zyme is not commercially available, elastase-inhibitory activity is routinely measured using porcine pancreatic elastase. The proteinase used in the assay must be free of other proteolytic enzymes, because oq-PI would also bind to such impurities and would, therefore, give incorrect values. Additionally, cysteiny117 and metalloproteinases ~ have been shown to inactivate a~-PI catalytically.

Trypsin-Inhibitory Assay Reagents Buffer: 0.01 M Tris-HC1, pH 8.0 Substrate: Benzoyl-L-arginine ethyl ester (Mann), 0.58 mM. Dissolve 20 mg of the hydrochloride salt in 100 ml of the Tris-HC1 buffer. Prepare fresh daily. Enzyme: Porcine trypsin. Prepare a stock solution of 1 mg enzyme in 5 ml of 2 mM HC1. This solution is stable at 4°C for at least 2 weeks. Procedm'e. Porcine trypsin (usually 0.10 ml) is dispensed into a series of 13 x 100-mm tubes, together with 0.2 ml of buffer. The inhibitor solution (up to 0.2 ml) is added and the mixture incubated at room temperature for at least 1 min. The assay is begun by adding substrate solution to a final volume of 3.0 ml. After mixing well, the sample is transferred to a 3.0-ml cuvette and the increase in absorbance at 253 nm measured for 1 min. The trypsin control, without inhibitor, should have a ~A253 of 0.1-0.2 per minute. The amount of ~I-PI used should be adjusted to give a ~A2~3 between 30 and 70% of the control. One unit of trypsin-inhibitory activity is defined as that amount which reduces the 5A253 of trypsin by 1.0 absorbance units per minute relative to the trypsin control. The value of ~ (change in molar extinction coefficient) for the complete hydrolysis of substrate is 1160. Therefore, a change in absorbance of 1.0 unit translates into the production of 2.59 ~zmol of product.

Elastase InhibitoiT Activity Reagents B,ffer: 0.2 M Tris-HC1, pH 8.0 Substrate: Succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide(Sigma). Dissolve 45 mg in 5 ml of dimethylformamide or dimethyl sulfoxide to yield a 20 mM stock solution that is stable at 4°C for several weeks. ~; D. Johnson and J. Travis, Biochem. J. 163, 639 (1977). ~" J. Morihara, T. Suzuki, and K. Oda, Inject. lmmun. 24, 188 (1979).

[55]

HUMAN ~I-PROTE1NASEINHIBITOR

757

Enzyme: Porcine pancreatic elastase, chromatographically purified

(Worthington). Prepare a stock solution of 0.3 mg e n z y m e in 5 ml of 0.1 M Tris-HC1, pH 8.0. The enzyme is not stable below pH 4.0. Procedure. Porcine elastase (usually 0.5 ml) is dispensed into 13 × 100-mm tubes and 0.2 ml of buffer is added. Inhibitor solution (up to 0.2 ml) is mixed with the elastase and the mixture incubated at room temperature for at least 1 min. Buffer is added for a total of 2.95 ml and the assay is begun by adding 0.05-ml of substrate solution. After mixing well, the sample is transferred to a 3.0-ml cuvette and the increase in absorbance at 410 nm measured for 1 rain. A ~A410 per minute for the elastase control should be 0.1-0.2. The amount o f a r P I used in the assay should reduce the ~A410 by 30-70%, relative to the control. One unit of elastaseinhibitory activity is defined as the reduction in the ~A4~0 o f the elastase control by 1.0 absorbance unit per minute. Since the molar extinction coefficient ofp-nitroaniline is 8800 at 410 nm, one unit corresponds to the production of 0.341 ~mol o f product. Purification Procedure The purification described here is a modification of the original procedure described by Pannell et al. in 1974, ~ and primarily involves removal of albumin from plasma by affinity chromatography on dye-bound gels. Preparation o f Cibacron Blue Sepharose

The coupling of Cibacron Blue F-3GA (Ciba-Geigy) to Sepharose follows the procedure of Bohme et al. 1.~The coupling of the dye to Sepharose requires heating to 95°C. For this reason a cross-linked gel stable to high temperatures is used. 2° Typically, 1 liter of washed Sepharose-4B or 6B is mixed at room temperature with 1 liter of 1 M N a O H , containing 5 g of NaBH4. To this mixture is added, with stirring, 20 ml of epichlorohydrin and the mixture heated to 60°C for 1 hr. The cross-linked gel is then washed on a coarse Buchner filter with hot water until the washings are neutral. The S e p h a r o s e - d y e conjugate referred to as Cibacron Blue Sepharose is prepared by suspending 500 ml of cross-linked gel in an equal volume of water and warming the mixture to 60°C. The dye (5 g in 50 ml of water) is added dropwise with vigorous stirring and, after 15 rain, 50 g of NaC1 is added. The mixture is heated to 95°C to ensure efficient coupling, and 10 g t' H. J. Bohme, G. Kopperschlagaer, J. Schultz, and E. Hofmann, J. Chromatogr. 69, 209 (1972). ~ J. Porath, J. C. Janson, and T. Lass, J. Chromatogr. 60, 167 (1971).

758

INH1BITORS OF VARIOUS SPECIFICITIES

[55]

of Na2CO3 is added. After 30 min, the dyed gel is washed with hot water, followed by 0.05 M Tris-HCl-0.05 M NaCI (pH 8.0), until the washings are color free. Traces of blue dye may still elute during the first passage of plasma through columns of this material. However, subsequent usage usually indicates no further dye leakage. Gel prepared in this manner is capable of binding up to 40 mg of human albumin per milliliter of dye-gel conjugate. Inhibitor Purification Step 1. Ammonium Su!['ate Fractionation. Solid (NH4)zSO4 is added with stirring to human plasma (usually 1 liter) to bring the concentration to 0.5 saturation (213 g per liter of plasma). The solution is clarified by centrifugation at 25,000 g (10 rain at 4°C), and the precipitate discarded. (NH4)2804 is then added to bring the concentration to 0.8 saturation (210 g/liter). After centrifugation the precipitate is retained, dissolved in 100 ml of 0.03 M sodium phosphate buffer (pH 6.7), and dialyzed against the same buffer for 24 hr (4 liters of buffer, 4°C, four changes). Step 2. Cibacro, Bhte Sepharose Fractionation. A column of Cibacron Blue Sepharose is prepared (5.0 × 100 cm) and equilibrated with 0.03 M sodium phosphate buffer, pH 6.7. The yellow solution obtained from step 1 is applied to the column. The column is then washed with equilibration buffer (225 ml/hr) and 19-ml fractions collected. The fractions containing ~I-PI are eluted in the void volume, together with transferrin, orosomucoid, pre-albumin, and traces of high-molecular-weight components. Collection of fractions may be discontinued when the A280 falls below 0.100. Since albumin remains tightly bound to the column (the color of the dye-albumin complex becomes a light blue), stripping of the protein may be obtained by elution with equilibration buffer containing 0.50 M KSCN. The eluted albumin is usually about 95% pure and contains only traces of albumin dimer. Reequilibration of the column with starting buffer makes it suitable for further use. Occasionally, however, the column should be further washed with either 6 M urea or 5 M guanidine-HCl to remove tightly bound components, such as lipoproteins, before reequilibration. In our laboratory this is usually performed when the binding capacity of the column for albumin is reduced to less than 20 mg albumin/ml gel conjugate. Step 3. Frac'tionation on DEAE-Celhdose. A column of DEAEcellulose (DE-52, Whatman Chemicals) is prepared (2.5 × 30 cm) and equilibrated with 0.03 M sodium phosphate buffer, pH 6.5. The pooled fractions containing cq-P1, obtained in step 2, are adjusted to pH 6.5 with 0.01 N HCI and charged directly onto the column. The column is washed

[55]

HUMAN O~I-PROTEINASE INHIBITOR

759

1.6f

E

14

¢0

i

1.2

cO (M

1.0

b..I 0 Z o,8

-

e

1313 t'r- 0.6 0

4,o

io--

Or)

.

0,4

40

80

120

160

200

240

280

320

360

400

440

,-;-

480

FRACTION NUMBER FIG. 1. Salt gradient-elution c h r o m a t o g r a p h y of human cq-proteinase inhibitor on D E A E - c e l h d o s e . Q - - Q , Absorbance at 280 rim: A - - A , inhibitory activity; ...... , NaCI concentration. Arro w represents initiation of gradient.

with equilibration buffer until the A280 is less than 0.050. An inactive breakthrough peak containing transferrin is usually observed. A linear gradient of NaC1 from 0 to 0.2 M NaC1 in 2000 ml of 0.03 M sodium phosphate buffer (pH 6.5) is applied to develop the chromatogram (30 ml/hr) and 5-ml fractions collected. Three peaks are obtained by this procedure (Fig. 1). The first contains all of the u r P I , whereas the last two are orosomucoid (~l-acid glycoprotein) and pre-albumin, both of which are obtained in homogeneous form. Step 4. Gel-Filtration C/n'onum~,raphy. A column of Sephadex G-75 (5.0 × 100 cm) is equilibrated with 0.05 M Tris-HCI-0.05 M NaCI, pH 8.0. The pooled active fractions from step 4 are adjusted to pH 8.0 with 1 M Tris (unbuffered) and concentrated to 30 ml by ultrafiltration on a UM-10 membrane. After application to the Sephadex G-75 column, equilibration buffer is used to elute the oq-PI containing fractions (flow rate 30 ml/hr; 5-ml fractions). Inactive protein appears in the void volume, followed by the purified inhibitor. Normally, ch-PI is stored frozen in the equilibration buffer, at concentrations up to 5 mg/ml, without loss of inhibitory activity.

760

INHIBITORS OF VARIOUS SPECIFICITIES

4

[55]

5

FIG. 2. Polyacrylamide-gelelectrophoresis of human cq-proteinase inhibitor at each stage of purification. (I) Whole human plasma; (2) (NH4)2SO4 fractionation; (3) Cibacron Blue Sepharose chromatography; (4) DEAE-cellulose chromatography; (5) gel-filtration chromatography.

Figure 2 depicts the purification as noted by gel electrophoresis of fractions obtained in the steps given above. Table I indicates the r e c o v e r y at each stage of the purification. Properties

Stability H u m a n oq-PI is stable for several months at 4°C in solutions containing 0.01% sodium azide. This protection is required because of the potential inactivating effect of proteinases released from bacterial contaminants. O~l-PI m a y also be stored frozen in solution at - 80°C, but once thawed the protein should not be refrozen because this leads to inactivation. Lyophilized preparations of al-PI, dialyzed free of salts before freezedrying, retain less than half of their activity on solubilization. H o w e v e r , preparations lyophilized in the presence of 0.15 M NaC1 retain nearly all of their inhibitory activity after resolubilization.

[55]

761

HUMAN O~I-PROTEINASE INHIBITOR TABLE I PURIFICATION OF O!I-PROTEINASE INHIBITOR

Step 1. 2. 3. 4. 5.

Starting plasma (850 ml) (NH4)2SO4, (0.5-0.8 saturation) Cibacron Blue Sepharose DEAE-cellulose Sephadex G-75

Protein (mg)

Activity units (total)

Specific activity (units/ A2s0,,m)

Recovery (%)

Purification (-fold)

54,570 24,720 2332 927 350

4529 4252 4012 2808 2793

0.083 0.172 1.72 3.03 7.98

100 94 89 62 61

1 2.1 20.7 36.5 96

Unlike many of the low-molecular-weight inhibitors, oq-PI is not stable below pH 5.5. This is probably because inactive polymers form near the isoelectric point. However, rapid acidification to pH 2.0 does not seem to cause irreversible inactivation of the inhibitor, since readjustment to neutral pH results in the retention of most of the inhibitory activity. '1

PmiO, The purified inhibitor exhibits a single band on gel electrophoresis at alkaline pH, as well as on electrophoresis after treatment with sodium dodecyl sulfate (reduced and nonreduced preparations). Two major and three minor bands are observed after isoelectric focusing of the PiM proteins in a pH 4-6 ampholyte system. It is believed that these differences are due to different degrees of sialylation of the individual protein moieties, since treatment of the purified preparation with neuraminidase shifts the pattern to focus at a higher pH value as one major fraction, s Immunoelectrophoresis against either anti-whole human plasma or antihuman al-PI gives only single precipitin lines in each case. Ouchterlony immunodiffusion against antibodies to albumin, orosomucoid, al-antichymotrypsin, antithrombin III, pre-albumin, group-specific components, and transferrin are all negative. Such immunological tests are particularly important in the case of orosomucoid, since, due to its high carbohydrate content, common protein stains are not tightly bound and are removed during prolonged destaining. Physical Properties Human al-PI is a glycoprotein containing about 12% carbohydrate. It has MW 53,000 determined by both sedimentation equilibrium experi~ C. Glaser, L. Karic, and A. Cohen, Biochim. Biophys. Acta 491, 325 (1977).

762

INHIBITORS OF VARIOUS SPECIFICITIES

[55]

ments and gel electrophoresis after treatment with sodium dodecyl sulfate. "'~2 The protein has an e~g of 5.30 ~:''-' and a range of isoelectric points from 4.4 to 4.8, depending on the phenotype. The determination of phenotype by isoelectric focusing has been clearly shown to be a valuable analytical tool. = Human c~i-PI has the following amino acid composition: LysagHis~aArgrTrplAsp49Thr25SerzaGlu~4Pro2zGlyz4Ala24 ~Cys~Val2rMetsIleisLe usoTyrGPh%8 The single cysteine residue is usually intermolecularly linked to either cysteine or glutathione. 24 The amino-terminal sequence of oq-PI is as follov/s: Glu-Asp-Pro-Glu-Gly-Asp-Ala-Ala-Gln-Lys-Thr-Asp-Thr-Ser His-His-Asp-Gin-Asp- His-Pro-Thr-Phe-Asn- Lys- Ile-Thr-ProAsn-Leu-Ala-Glu-Phe-Ala-Phe-Ser-Leu-Tyr-Arg-Gln-Leu-Ala Val-Thr-Gly ~ The carboxy-terminal sequence has also been determined: Gly-Lys-Val-Val-Asn-Pro-Thr-Gln- Lys 26 However, although many other fragments have been isolated and sequenced, the complete primary structure of ~-PI has not yet been determined."r-a, The carbohydrate composition of c~i-PI is as follows: N-acetyl hexosamine~2hexoseissialic acidr, s The sequence of the carbohydrate linked to asparaginyl residues of ~I-PI through N-glycosidic linkages has been reported, a~'a' Three side chains were found, two of type A and one of type B. The A-chain composition consisted of ManaGala(GlcNAc)4 and (NeuAc)z, while the B chain contained ManaGal~(GlcNAc)s and (NeuAc)a. A and B have been found to be identical except that B contains an additional mannose-linked trisaccharide. 2'-' M. Schonenberger, Z. NatttCfi~rsch., B." Am~rg. Chent., Org. Chem., Biochem., Biophy.~., Biol. B10, 474 (1955). ~a j. Pierce, J. O. Jeppson, and C. B. Laurell, Anal. Biochem. 74, 227 (1976). ~ C. B. Laurell, J. A. Pierce, U. Persson, and E. Thulin, Eur. J. Biochem. 57, 107 (1975). '-':' J. Travis, D. Garner, and J. Bowen, Biochenli.strv 17, 5647 (1978). ~; J. Travis and D. Johnson, Biochem. Biophys. Res. C o m m u n . 84, 219 (1978). ..,7 W. Hrong and J. C. Gan, Int. J. Biochem. 6, 555 (1975). ~ M. C. Owen, M. Lorier, and R. W. Carrell, FEBS Lett. 88, 234 (1978). ~" D. J o h n s o n and J. Travis, J. Biol. Chem. 253, 7142 (1978). :~o R. W. Carrell, M. C. Owen, S. O. Brennan, and L. Vaughan, Biochem. Biophys. Res. C o m m u n . 91, 1032 (1979). a~ L. Hodges, R. Laine, and S. K. Chan, J. Biol. Chem. 254, 8208 (1979). a~ T. Mega, E. Lujan, and A. Yoshida, J. Biol. Chem. 255, 4057 (1980).

[55]

HUMAN RI-PROTEINASE INHIBITOR

763

Biological Properties Speci[icity

Human ~ - P I specifically inhibits serine proteinases, including pancreatic and leukocyte elastases, 3:~-:~ pancreatic trypsin and chymotrypsin, :~3 leukocyte cathepsin G, ~" thrombin, :~¢ plasmin, 3~ acrosin, 39 kallikrein, 4° and skin and synovial collagenases. 4~'4' In addition to these interactions, it has also been reported that microbial enzymes from Aspergilhls 4:~ and B. snbtilus 44 are also inactivated by a r P I . All of these interactions appear to be due to the formation of a I : 1 molar complex that is stable to acid, as well as to boiling in 1% sodium dodecyl sulfate. Significantly, a~-PI does not inhibit nonserine proteinases. In fact, the inhibitor is rapidly inactivated by papain and cathepsin B (thiol proteinases), as well as by an elastolytic enzyme from P s e u d o m o n a s aeroginosa (a metalloproteinase)? r'~ Structural studies indicate that this reaction is occurring by peptide-bond cleavage at the reactive site. Kinetic Properties The association rate of enzymes with ax-P[ has been determined for a number of serine proteinases. ~ Human leukocyte elastase was found to have the highest/,a~ (6.5 × 107), followed by human chymotrypsin (5.9 × 10~), human leukocyte cathepsin G (4.1 × 10~), human anionic trypsin (7.3 × 10~), human cationic trypsin (1.1 × 104), human plasmin (1.9 × 10"), and human thrombin (4.8 × 10~). It would appear from these data that the primary function of a~-PI is to control the activity of leukocyte elastase and not trypsin, as the original name given for this protein tended to indicate. Significantly, chemical oxidation of c~-PI markedly reduces

:~:~H. C. Schwick, N. Heimburger, and H. Haupt, Z. G e s a m t e Inn. Med. lhre G r e , z g e b . 21, 1 (1966). :~4 A. Janoff, A m . Rev. Respir. Dis. 105, 121 (1972). :~; P. D. Kaplan, C. K u h n , and J. A. Pierce, ./. Lab. Clin. :$led. 82, 349 (1973). :~'~R. Baugh and J. Travis, Biochemisn3 15, 836 (1976). :~7 N. R. Matheson and J. Travis, Biochem. J. 159, 495 (1976). :~ A. Rimon, Y. S h a m a s h , and B. Shapiro, J. Biol. Chem. 241, 5102 (1966). :~' H. Fritz, N. Heimburger, M. Meier, M. Arnold, L. J. D. Zaneveld, and G. B. F. S c h u m a c e r , Hoppe-Seyler's Z. Phv.siol. Chem. 353, 1953 (1972). ~" H. Fritz, B. Brey, A. Schmal, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 350, 1551 (1969). ~ Y. Tokoro, A. Z. Eisen, and J. T. Jeffrey, Biochim. Biophys. Acta 258, 289 (1972). 'e E. D. Harris, D. R. DiBona, and S. M. Krane, J. Clin. lnve.~t. 48, 2104 (1969). ~:~R. Bergkvist, Acta Chem. Scaml. 17, 2239 (1963). 44 V. Wicher and J. Dolovich, lmmum~chemistry 10, 239 (1973).

764

INHIBITORS OF VARIOUS SPECIFICITIES

[55]

TABLE II COMPARATIVE AMINO ACID SEQUENCES AT THE REACTIVE CENTER OF PROTEINASE INHIBITORS

cq-Pl" Antithrombin III ~' Garden bean (elastase) '~ Lima bean (trypsin)~ Lima bean (chymotrypsin)j Soybean (trypsin)~ Soybean (chymotrypsin) ~'

Leu-Glu-Ala- lie- Pro-Met- Ser- lie- Pro- Pro-Glu- Val-Lys Val- Val- Ile- Ala- Gly- Arg- Set" ~-Leu- Asn- Pro-Asn Val-Cys- Thr- Ala- Set- lie- Pro-Pro- Gln-Cys-lle His-Cys-Ala-Cys- Thr- Lys- Set- lie- Pro-Pro- Gln-Cys-Arg Ser-Cys- Ile-Cys-Thr- Phe- Set- lie- Pro- Ala-Gln-Cys-Val Gln-Cys-Ala-Cys- Thr- Lys- Set- Asn- Pro- Pro- Gln-Cys-Arg Ser-Cys- lle-Cys- Ala- Leu- Ser- Tyr- Pro- Ala- Gln-Cys-Arg P6 Ps P4 Pa P2 P1 P~ P~ P~ P~ P~ P~ P~

" D. Johnson and J. Travis, J. Biol. Chem. 253, 7142 (1978). H. Jornvall, W. W. Fish, and 1. Bjork, FEBS Lett. 106, 358 (1979). ~ Italicized residues are homologous to those in arPI. ,l K. A. Wilson and M. Laskowski, St., Bayer Symp. 5, 344 (1974). " F. C. Stevens, C. Wuerz, and J. Krahn, Bayer Syrup. 5, 344 (1974). J. Krahn and F. C. Stevens, Biochelnistry II, 1804 (1970). ~' S. Odani and T. Ikenaka, J. Biochem. (Tokyo) 71, 839 (1972). h T. Ikenaka, S. Odani, and T. Koide, Bayer Syrup. 5, 325 (1974). the ka~s for l e u k o c y t e elastase (to 3.1 × 104) a n d for p a n c r e a t i c e l a s t a s e (to zero). Reactive Center T h e a m i n o acid s e q u e n c e s u r r o u n d i n g the i n h i b i t o r y site o f a r P I has b e e n d e t e r m i n e d . This w a s partly o b t a i n e d b y f o r m i n g c o m p l e x e s o f a l - P I with v a r i o u s serine p r o t e i n a s e s , d i s s o c i a t i n g the c o m p l e x e s with n u c l e o philic reagents, a n d e x a m i n i n g n e w a m i n o - t e r m i n a l s e q u e n c e s d e r i v e d by p r o t e i n a s e c l e a v a g e of the a l - P I m o l e c u l e d u r i n g the d i s s o c i a t i o n ? 5 With all e n z y m e s s t u d i e d ( t r y p s i n , c h y m o t r y p s i n , elastase), the n e w a m i n o - t e r m i n a l s e q u e n c e s o b t a i n e d w e r e identical b u t different from that of the n a t i v e inhibitor. Significantly, p a p a i n c l e a v e d a r P I at the same site, e v e n t h o u g h no i n h i b i t i o n o f this e n z y m e w a s f o u n d . F u r t h e r studies inv o l v i n g p a p a i n c l e a v a g e o f d e n a t u r e d a r P I r e s u l t e d in the isolation a n d s e q u e n c i n g o f a p e p t i d e w h o s e s t r u c t u r e o v e r l a p p e d with that o f the n e w s e q u e n c e d e r i v e d b y c o m p l e x dissociation. '9 This s e q u e n c e of a m i n o acids, t o g e t h e r with s e q u e n c e s d e r i v e d for the r e a c t i v e site o f o t h e r s e r i n e p r o t e i n a s e inhibitors, is given in Table II. H o m o l o g y with o t h e r i n h i b i t o r s is a p p a r e n t . T h e r e h a v e b e e n claims that a r P I m a y h a v e m o r e t h a n one i n h i b i t o r y site and that, in fact, it m a y be a " m u l t i h e a d e d " inhibitor. 4~-49 This re4~D. Johnson and J. Travis, Biochem. Biophys. Res. Commun. 72, 33 (1976). 4, T. Lo, A. B. Cohen, and H. James, Biochim. Biophys. Acta 453, 344 (1976).

t56]

HUMAN0q-ANTICHYMOTRYPSIN

765

mains to be unequivocally proved, however, since nonspecific peptidebond cleavage during the interaction of proteinases with a r P I may be responsible for the interpretations drawn by these authors. Inactivators a~-PI inhibitory activity is readily abolished by interaction with several nonserine proteinases, tT'~°'~ It is also sensitive to both chemical and enzymatic oxidants, ~ ~ which convert the reactive-site methionine to a sulfoxide form. Cigarette smoke also inactivates al-PI, apparently by the same oxidation process. ~ It has been suggested in all of these studies that the oxidation process may occur in vivo, resulting in the loss of inhibitory activity in the lung and the concomitant development of emphysema due to uncontrolled proteolysis by proteinases released from phagocytic cells. ~7 j. Baumstark, C. Lee, and R. Luby, Biochim. Biophys. Acta 484, 400 (1973). ~'~ H. James and A. B. Cohen, ,I. Clin. Invest. 62, 1344 (1978). ~ S. Satoh. T. Kurecki, L. Kress, and M. Laskowski, Sr., Biochem. Biophys. Re,s. Cornmum 86, 130 (1979). ;'" R. W. Moskowitz and C. Heinrich, J. Lab. Clin. Med. 77, 777 (1971). .-,i L. F. Kress and E. A. Paroski, Biochem. Biophys. Res. Commun. 83, 649 (1978). ~e D. Johnson and J. Travis, J. Biol. Chem. 254, 4022 (1979). :':~ N. R. Matheson and J. Travis, Biochem. Biophys. Re.s. Commun. 88, 402 (1979). ~4 H. Carp and A. Janoff, J. Clin. Invest. 63, 5461 (1979). :':' D. Johnson, Am. Rev. Respir. l)is. 121, 1031 (1980). :'~ H. Carp and A. Janoff, Am. Rel'. Respir. Dis. 118, 617 (1978).

[56]

Human arAntichymotrypsin

B y JAMES T R A V I S a n d M I T S U Y O S H I M O R I I

Human oq-antichymotrypsin (al-Achy) is a plasma proteinase inhibitor that specifically inactivates serine proteinases of the chymotrypsin class. This protein was first described by Schultze et al. as al-X glycoprotein ~ but was later found to be a chymotrypsin inhibitor. ~ It is now known to specifically control the activity of chymotrypsin-like proteinases from phagocytic cells (neutrophils, basophils, tissue mast cells), including cathepsin G and "chymase."3"4 Furthermore, it possesses the unusual feaH. E. Schultze, K. Heide, and H. Haupt, Klin. Wochenschr. 40, 427 (1962). N. Heimburger and H. Haupt, Clin. Chim. Acta 12, 116 (1965). :* J. Travis, J. Bowen, and R. Baugh, BiochemLstrv 17, 5652 (1978). K. Beatty. J. Bieth, and J. Travis, .l. Biol. Chem. 255, 3931 (1980).

METHODS IN ENZYMOLOGY.VOL. g0

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