190, No. 1, September,
COMMUNICATIONS The Inhibition
of Human Leukocyte
by Basic Pancreatic
Bovine basic pancreatic trypsin inhibitor inhibits human leukocyte elastase with a dissociation constant as low as 1 pM at pH 8.0 under appropriate conditions. The affinity increases with pH and ionic strength. Trypsin, chymotrypsin, and elastase are bound at the same site of the inhibitor. Prior binding of elastase to plasma as-macroglobulin leads to a lo-fold decrease of its affinity for the inhibitor. The basic inhibitor from bovine pancreas is a “microprotein” (M, = 6500) which forms very stable complexes with pancreatic kallikrein, trypsin or chymotrypsin (I-3) but is ineffective on pancreatic elastase (4). It seems also to be a very poor inhibitor of human leukocyte elastase (5-7). Its binding constant for the latter enzyme has never been determined and reports concerning its inhibitory potency are rather conflicting. For instance, Starkey and Barrett (7) found 65% inhibition with a 80 pM concentration of inhibitor, Kruze et al. (6) obtained only 68% inhibition with a 1 mru concentration of this protein and Schiessler et al. (8) believe that the pancreatic inhibitor does not inhibit leukocyte elastase at all. The Sepharosebound inhibitor was, however, used by Baugh and Travis (9) for the “affinity” chromatographic isolation of human leukocyte elastase. In view of these conflicting results, it became important to reinvestigate the elastase-inhibitor interaction and to decide whether Sepharose-bound inhibitor acts really as an affinity absorbent. Human leukocyte elastase was isolated by the method of Baugh and Travis (9). Basic pancreatic trypsin inhibitor was from Choay Laboratories, Paris. The source and active site Litrations of bovine trypsin and cY-chymotrypsin as well as the isolation of human plasma a*-macroglobulin have been described elsewhere (10). Elastase activity was measured with succinyl-(L-alanine)a-p-nitroanilide (11). The inhibitor, the substrate and an-macroglobulin were dissolved in the appropriate buffers, whereas the stock solution of enzyme (0.32 mg/ml) in 1 mu CH&OOH was used directly. To determine the dissociation constants of free elastase, the mixtures formed of increasing amounts of inhibitor and constant amounts of enzyme (0.2-l PM) were incubated during 5 min at 25°C before addition of substrate. The dissociation constant of a2macroglobulin bound elastase was measured by reacting elastase (0.21 PM) with an excess of az-macroglobulin (1.5 PM) during 10 min at 25°C. After subsequent addition of inhibitor, the mixtures were incubated for 30 min at 25°C before addition of substrate. In all cases two substrate concentrations (0.5 mu and 2 mM) were used. The other technical details are given in the results section. When elastase (0.21 pM) and inhibitor (18 pM) were 358 0003-9861/78/1901-0358$02.00/O Copyright All rights
0 1978 by Academic Press, of reproduction in any form
reacted at pH 8.0 and 25°C for various periods of time before addition of substrate, the extent of inhibition (76%) did not change with time. On the other hand, the same percentage of inhibition was obtained whether the substrate was added to the enzyme + inhibitor mixture or the enzyme was added to the substrate + inhibitor mixture. These experiments demonstrate that the equilibrium between enzyme, inhibitor, substrate and their respective complexes is reached within the time required to mix the reagents. Figure 1 shows the effect of increasing amounts of inhibitor on the activity of constant amounts of elastase tested with two concentrations of substrate. The shape of the inhibition curves indicates that the binding is not very tight. On the other hand, the extent of inhibition decreases with the substrate concentration suggesting competitive inhibition. With a large excess of inhibitor (0.57 mM) the inhibition was complete. The enzyme-inhibitor complex does therefore not exhibit a residual enzyme activity in contrast to what has been observed for the chymotrypsin-pancreatic inhibitor complex (3). Since a molar excess of inhibitor over enzyme of at least 15 is required to observe appreciable inhibition, the concentration of bound inhibitor may be neglected with respect to the total concentration of inhibitor. Hence the graphical method of Dixon (12) could be used to determine K, (see the insert of the figure). A Cornish-Bowden plot (13) confirmed that the inhibition is purely competitive. The value of K, (3.5 pM) shows that bovine pancreatic inhibitor is a moderately potent inhibitor of human leukocyte elastase but is far from being inactive on this enzyme as claimed by others (8). It is however much more potent on trypsin (K, = 0.06 PM, Ref. 2) on a-chymotrypsin (K, = 9 no, Ref. 3) and on kallikrein (K, = 1 no, Ref. 1) under similar conditions. This binding constant is nevertheless sufficiently low for affinity chromatography (14). Since one of the aims of this investigation was to check whether Sepharose-bound inhibitor (9) was really acting as an affinity adsorbent, we have measured the K, with the buffers used by Baugh and Travis to isolate leukocyte elastase (9). These authors used buffer 2 (Table I) to adsorb elastase on the affinity column and buffers 3 and 5 to wash out impurities. The corresponding three values of K, are still
ery of elastase activity in the course of the dissociation experiment. Plasma as-macroglobulin is able to form enzymatitally active complexes with proteolytic enzymes. The trypsin-macroglobulin complex can be inhibited by pancreatic inhibitor but the K, (2 pM) is 3 X 10’ times higher than that of free trypsin (16). Since the dissociation constant of the elastase-pancreatic inhibitor TABLE
DISSOCIATION CONSTANTS (K,) OF THE COMPLEXES FORMED OF BASIC PANCREATIC TRYPSIN INHIBITOR AND FREE OR ~~~~~~~~~~~~~~~~~~~~~~ HUMAN LEUKOCYTE ELASTASE AT 25°C Buffer
A. Free Elastin of reaction medium
FIG. 1. Inhibition of human leukocyte elastase (0.21 pM) by basic pancreatic trypsin inhibitor at pH 8.0 (0.2 M Tris-HCl) and 25°C. The substrate concentrations are 0.5 mu (0) and 2 mu (0). The insert is a replot of the data according to Dixon (12). The rate V is in arbitrary units. sufficiently low to allow elastase to be bound to the column. With buffer 7 used to elute elastase, a marked increase in K, is observed (Table I). It may therefore be concluded that affinity chromatography is achieved in the procedure of Baugh and Travis (9). The magnitude of the increase of K, between pH 8.0 and 5.0 is of the same order as that reported for the K, of the chymotrypsin-pancreatic inhibitor complex (3). At pH 8.0 we noticed that K, is strongly dependent upon ionic strength. This led us to investigate also the influence of ionic strength at pH 6.5 and 5.0. As shown in Table I, high salt concentrations decrease K, whatever the pH. This suggests that hydrophobic interactions play an important role in the binding of leukocyte elastase to pancreatic inhibitor. The importance of hydrophobic contacts between this inhibitor and trypsin and chymotrypsin has also been inferred from thermodynamic (2, 3) and crystallographic (15) investigations. Trypsin and chymotrypsin compete for the same binding site of pancreatic inhibitor (2, 3). This active center is also responsible for the binding of leukocyte elastase, as shown by the competition experiments reported in Table II. Chymotrypsin displaces elastase from its complex with the inhibitor and the chymotrypsin-inhibitor complex does not inhibit elastase. The results are not clear-cut in the case of trypsin because this enzyme “inhibits” elastase as shown by separate experiments: for instance a mixture of 0.53 ,UM elastase and 8 pM trypsin is only half as active as elastase alone. This effect accounts for the low recov-
1 2 3 4 5 6 7
Tris 50 mu Tris + 50 mM NaCl 50 mM Tris + 400 mM NaCl 50 mM phosphate 50 mu phosphate + 400 mM NaCl 50 mM acetate 50 mu acetate + 400 mM NaCl
B. a*-Macroglobulin-Elastase 1
“The Tris HCl. bA mixture NazHPOd. ‘A mixture CHGOOH.
of 50 mM TABLE
8.0 with 50
COMPETITION BETWEEN BOVINE TRYPSIN AND aCHYMOTRYPSIN AND HUMAN LEUKOCYTE ELASTASE FOR THE BINDING OF BASIC PANCREATIC TRYPSIN INHIBITOV Composition
0.53 PM elastase 0.53 PM elastase + 8 pru inhibitor 0.53 pM elastase + 8 pM inhibitor + 8 PM trypsin* 8 FM trypsin + 8 w inhibitor + 0.53 pM elastase 0.53 PM elastase chymotrypsinb 8 PM chymotrypsin /.lM elastase
+ 8 pM inhibitor + 8 pM inhibitor
+ 8 pM
a The assays were performed in 0.2 M Tris-HCl buffer, pH 8, at 25°C with 0.5 mu substrate. ’ Concentration of active sites.
complex is already in the micromolar range, it was thought that macroglobulin-bound elastase would be completely resistant to inhibition. This was not the case since the K; (33 pM) is only 10 times higher than for free elastase. Investigation of the time dependency of the inhibition showed however that equilibrium was reached only after 15 min of incubation. During these experiments we were able to confirm the lo-fold activation of elastase activity by Lua-macroglobulin (17). REFERENCES 1. FRITZ, H., SCHULT, H., MEISTER, R., AND WERLE, E. (1969) 2. Physiol. Chem. 350, 1531-1540. 2. VINCENT, J. P., AND LAZDUNSKI, M. (1972) Biochemistry 11, 2967-2977. 3. VINCENT, J. P., AND LAZDUNSKI, M. (1973) Eur.
J. Biochem. 38,365-372. 4. PLOTTER, J.,
9. BAUGH, R. J., AND TRAVIS, J. (1976) 15,836~841. 10. AUBRY, M., AND BIETH, J. (1976)
phys. Acta 438,221-230. 11. BIETH,
J., SPIESS, B., AND WERMUTH,
C. G. (1974)
Biochem. Med. 11,350-357. 12. DIXON, M. (1953) Biochem. J. 55, 170-173. 13. CORNISH-BOWDEN, A. (1974) Biochem. J. 137, 143-144. 14. CUATRECASAS, P., AND ANFINSEN, C. B. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 345-378, Academic Press, New York. 15. BLOW, D. M., AND WRIGHT, C. S. (1972) J. Mol. Biol. 69, 137-144. 16. JACQUOT-ARMAND, Y., AND KREBS, G. (1969) FEBS Lett. 4, 21-24. 17. TWUMASI, D. Y., LIENER, I. E., GALDSTONE, M., AND LEVYTSKA, V. (1977) Nature 267,61-63.
Biochim. Biophys. Acta 127,538~540. 5. JANOFF, A. (1977) in Pulmonary Emphysema and Proteolysis (Mittman, C., ed.), pp. 205-224, Academic Press, New York. 6. KRUZE, D., MENNINGER, H., FEHR, K., AND B~NI, A. (1976) Biochim. Biophys. Acta 438,503-513. 7. STARKEY, P. M., AND BARRETT, A. J. (1976) Biothem. J. 155.265-271. 8. SCHIESSLER, H., OHLSSON, K., OLSSON, I., ARNHOLD, N., BIRK, Y., AND FRITZ, H. (1977) 2.
Physiol. Chem. 358, 53-58.
PATRICK LESTIENNE JOSEPH G. BIETH
Laboratoire d’Enzymologie UER des Sciences Pharmaceutiques Vniversitk Louis Pasteur 3, rue de I’Argonne 67083 Strasbourg France Received April 14, 1978