ARCHIVES

OF BIOCHEMIS’IR\

A>\ID BIOt’H\rSICS

Purification

169,

91-101

(lg;:,,

and Preliminary

Characterization

Leukocyte JOSEPH Deportment

of Microbiolqq

C. TAYLOR’

of Human

Elastase’

ANI)

IRVING

, Scripps Clinic ond Rweorch

P. CRAWFORD Foundation.

Lo ./ol~o. C’oiifornio

920.97

Received .January 23. 197Z Affinity chromatography permits the purification of l-ii mg ot’ human leukocyte elastase f’rom the leukocytes contained in 500 ml of’ whole blood. Lysosomal granule proteins are extracted from polymorphonuclear leukocytes and subjected to chromatography on a column of’elastin-Sepharose. Contaminating proteins are eluted with bul’f’er containin:: 1 M NaCl and then elastase activity is eluted with hutt’er containing 8 hl urea. The enzyme retains all of’ its esterase activity against ,V-t-HOC-r.-alanine p-nitrophenyt ester alter exposure to 8 M urea and retams Z’f of its activity in the presence of 1’~ sodium dodecyl sulfate. In sodium dodecyl sulfate and 2.mercaptoethanol leukocyte elastase undergoes autolysis giving rise to several lo\v molecular weight Iragments. The molecular weight of the native enzyme is found to be 2?.000 t,y both gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresib. A characteristic set ot’ four isozymes is seen after acrylamide disc gel electrophoresis at pH -1..5.All bandh are active against elastin and also contain carboh?-drate by the periodic acid-Schif’f stain. On the basis of’ stain intensity. the slower mommy Isozymes appear to be richezt in carbohydrate. Active leukocyte elastase forms a complex with tu,-antltryphitl in a I:1 molar ratio. The elahtase must t,e enzymatically active for complex formation to occur.

Eriksson (1) observed that persons deficient in the major serum protease inhibtor, cu,-antitrypsin, frequently develop pulmonary emphysema at an early age. Since that time evidence has accumulated (2-S) implicating endogenous proteolytic enzymes as the major cause of the degradative changes in the lung found in advanced emphvsema. Of particular interest is the ability of canine and human polymorphonuclear (PMN13 leukocyte homogenates to induce experimental emphysema in dogs (2). Lysosomal granules from human PMN

leukocytes contain several proteases active at neutral or alkaline pH against a \rariet> of substrates (6-8). Methods have appeared which describe the purification of both an elastase and a collagenase (9-12). two leukocyte proteases which are potentially implicated in the loss of elasticity of lung tissue (13). Purified proteases are needed for studies involving their interaction with serum inhibitors and for biochemical characterization of the enzymes themselves. The major problems concerning isolation of human PMN leukocyte elastase have been the small amount of protein available in the starting material and the lability of’ the enzymatic activit! (9-l”). Because of this, difficulties arise in attempts to study the enzyme of persons from whom only limited amounts of’ blood may be taken (e.g., young. aged. or severely emphysematous persons). Although methods such as continuous flow filtration leukapheresis and continuous flow centrii’ugation are available to collect large nuni.

‘Supported in part by Grant:, GW1909X and HI,-1.5309 f’rom the National Institutes of’ Health. U.S. Public Health Service. ‘Postdoctoral fellow (PF-9101 of the American Cancer Society. ’ Abhrel-iations used in this paper: NBA. ,V-IBOC-[,sosomal Granules Leukocytes from normal individuals were isolated from whole blood as described previously (‘i). After hypotonic lysis of the red blood cells. the leukocytes were washed three times in 10 ‘LOvol of ice cold 0.34 M sucrose, sodium heparin added I 10.000 USP units/ml. 1 drop/l0 ml cell suspension, 1 * 10” cells/ml). and the cells lysed by repeated shearing through a 19. gauge needle. Once the Iysate had lost its vicosity the lysosomes were isolated by differential centrifugation at 1,999g and 17.OOOg. The 17,OOOg lysosomal pellet was stored at 20°C under O.O’L5M sodium pyrophosphate (pH 5.1) with lo’, sucrose. 1 M NaCl. and o.O:IF,M &alanine added.

Isolation

of

Leukoqte

Elastase

All steps in the isolation procedure were carried out at 4°C. Purif’ied Iyscwmal pellets were suspended in the same pyrophosphate buffer in which they were stored. then frozen and thawed three times. This procedure releases up to 90 9 of the available esterase activity. Following extraction, the material was centrifuged at 27.OOO~gand the supernatant added directly to a Sepharose-elastin column 10.9 4 cm) which had prr\ iolisly been equilibrated with 0.02;) 11

HUMAN

LEUKOCYTE

sodium pyrophosphate (pH 5.9) containing 10’; sucrose and 0.035 M @-alanine. The column was washed with 15-20 vol of the extraction buffer followed by 6 vol of 0.025 M sodium pyrophosphate, pH 6.4, containing 10% sucrose, 0.035 M p-alanine. and 8 M urea. The column flow rate was 55-60 ml/h. Active fractions which had eluted with the 8 M urea buffer were pooled, 10’; diluted 1:2 with 0.025 M sodium pyrophosphate. sucrose. and 0.035 M @-alanine, and then concentrated with 70% (NH,),SO,. Solid (NH,),SO, (4.72 g/10 ml) was added and the preparation stirred for 20 min. The precipitate was collected by centritugation (39,000~. 30 min) and dissolved in an amount of the supplemented pyrophosphate buffer so that the preparation contained greater than 12,000 units NRA esterase activity/ml. Purified HLE was stored at “0°C.

Polyacrylamide

Gel Electrophoresis

Electrophoresis of cationic proteins was carried out in 13? gels according to Reisfeld ct a/. (19. 201 omitting the urea. The gels were run at a constant current of 2.8 - 3 mA/gel tube for 1.5 h and stained for 30 min with 1’7r Naphthol blue black in 7.5”; acetic acid. Gels were destained with 7.5“; acetic acid. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis method used for the determination ot molecular weight was a modification of two different procedures. Acrylamide gels (lo?) and huffers were as described hy Laemmli (21). The samples were prepared for electrophoresis either by heating at 100°C for 1 h or incubating at 37°C for 2 h in 0.0125 ht sodium phosphate (pH 7.6) with 1’; dodecyl sulfate and 1’T 2mercaptoethanol (2%). Following this, an glycine equal volume of Tris (0.025 M) containing (0.192 M), dodecyl sulfate (O.lY I, glycerol (20’:). bromophenol blue (0.005%). and 10’; 2mercapto. ethanol was added and the mixture layered over the gel. Electrophoresis (with the anode in the lower reservoir) was performed at a constant current of :1 mA/tube. When the bromophenol blue dye was 2-3 cm from the bottom of the tube (approx 96 mini gels were removed, fixed in 50% acetic acid for 2 3 h (4°C). and stained overnight according to Weher and Osborn (22). Destaining (24 h) was accomplished in two changes of 30% methanol and 7.5’; acetic acid for :! and 3 h. 20? methanol and 7.5’; acetic acid for an additional :) h, and 10% methanol (7.5? acetic acid) overnight. The gels were stored in 7.5? acetic acid. Molecular weights of proteins were calculated as described previously (22) using chymotrypsinogen A. rihonuclease A (Pharmacia), trypsin, 0.chymotrypsin (Worthington), pepsin. and cytochrome c (Mann) as standards.

Sephadex G-7.5 Column

Chromatograph?

Sephadex G-75 was employed in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for molecular weight determinations. The

93

ELASTASE

and run at column (0.9 y 22 cm) was equilibrated 22°C with 0.025 M sodium pyrophosphate, pH 5.9, 0,035 bt fi-alanine. and O.O2”r sodium azide. Flow rate was approximately 30 ml/h Ovalbumin. chymotrypsinogen A. and ribonuclease A (Pharrnacia) were used as standards.

Detection

of Elastolytic Acyxflamide

Acticit,v GQ~,s

of HLE

in

Elastolytic activity 01 HLE was detected in 13’~ acnlamide gels by adding insoluble elastin ( 1.4’;. w/v) to gels prior to polymerization. This i> an adaption of the method originally described by Andary and Dabich (?:)I. Elastin was treated for 10 min in 0.05 ‘M TrissCl. pH 8.6, with a Hranson sonifier (power setting of .5) and the elastin allowed to settle for 1 h. Material not sedimenting during this period was collected by centrifugation ( 1.500~). washed and resuspended in the separatin, u ccl h buffer ol’ Reisfeld et al. ( 19. 20). Following electrophoresis. as described above, the gels were immersed in 0.X ~VI‘I’ris~Cl. pH 8.6, 0.035 ht p-alanine, and 0.2 M MgCI, for .T min. incubated 4-6 h at 37°C in a humidified chamber and observed for clearing of the gels.

Detection

PAS-Staining Material Acrylamide Gels

of

in

The method ot Kapitany and Zebrowski (24) was used for the staining ot carbohydrates on proteins following gel electrophoresis. Our only modification of their procedure was in the fixation of the proteins. Acid gels were fixed overnight and dodecyl sulfate gels in 50’; acetic acid. also overnight.

Neuraminidase

Treatment

of HLE

Neuraminidase ( Vibrio cholera, Calbiochem) was diluted from 500.000 mU/ml to 5,000 mU/ml with 0.05 M sodium acetate, pH 5.5. containing 7 rnM CaC’l,, lO$ sucrose, O.l,i M NaCI. and O.OYr sodium azide. A total of 150 mU 01’neuraminidase was added to 1X fig HLE in 200 ~1 of the diluent buffer. then incubated at :37”C for 24 h. Purit’ied autnlogous o,AT was obtained as described previously (25) and Was used as a control for the efficacy of the neuraminidaze treatment.

InactiL’ation

of

HLE rc,ith PMSF

PMSF (Pierce Chemical Co.) was added to purified HLE or to crude Iysosomal extracts at a final concentration of approximately :3.-t mr,t. After shaking in a 37°C water hath for 7 8 min the material was returned to an ice bath and activity measured. I’nder these conditions all of the NRA esterase activity was inactivated. Approximately 1.1 rn%f PMSF inhibited 685 of the enzyme acti\ it!

Protein L)eterminations Protein was determined by the method of Lowry et al. (26) using bovine serum albumin as standard.

94

TAYLOR

AND

CRAWFORD

trypsin-like protease of leukocyte lysosomes (12) seems unlikelv. Purificution of HLE The nature of the binding of the protein Table I presents the results of two typi- to Sepharose-elastin was examined. We cal purifications of leukocyte elastase. Ac- noted that a decrease in pH to 3.2 or an tivity eluting with 8 M urea usually repre- increase in salt concentration to 1 iv4NaCl sents 80-95”; of the total NBA esterase or 3 M (NH,),SO, eluted very little active activitv in crude lysosomal extracts. The enzyme. Urea at high molarity was found remaining NBA esterase activity in crude to elute all of the activity from the column. extracts passes through the Sepharose-elas- Factors in addition to active site interactin without binding and may be associated tion with substrate were implicated in the with a different protein. Activity eluting binding by experiments with PMSF-inacwith 8 M urea comprises approximately tivated HLE. Crude lysosomal extracts, 81% ofthe total elastolytic activity in crude treated with PMSF, were added to a Seextracts (data not shown). The 8 M urea pharose-elastin column, washed with eluate is the only column fraction capable buffer containing 1 M NaCl and eluted with of digesting elastin. Upon acrylamide gel 8 M urea exactly as described for active electrophoresis at pH 4.5 (Fig. 1). the preparations. Inactive HLE was bound to material eluted from the column by 8 M the Sepharose-elastin and the eluted prourea gives an isozyme pattern similar to tein was identical to active HLE in electrothat described by ,Janoff (9). Ohlsson and phoretic pattern and mobility (Fig. 1). Olsson (ll), and Schmidt and Havemann (12) for leukocyte elastase. These protein Properties of HLE bands all have the capability of digesting The isozyme pattern seen following elecelastin (Fig. 2). For convenience, however. trophoresis at pH 1.5 (Fig. 1) could be due we used NBA esterase activity, rather than to differences in charges of the proteins, to a method employing elastin as substrate. to measure leukocyte elastase in any given differences in the molecular weights. or to preparation. Purified preparations of HLE both charge and size differences. HLE strongly adheres to concanavalin Awere tested for activity against synthetic Sepharose (data not shown). This suggests substrates for trypsin and chymotrypsin. On a weight basis HLE is only half as that carbohydrate is present on the molecule. Because of this observation, we then active against N-Lu-benzoyl-Dr,-arginine-pHLE preparations for PASnitroanalide. a trypsin substrate, as por- examined staining material following acrvlamide gel cine pancreatic elastase or Lu-chymotrypsin (Worthington). HLE is ,500 times less ac- electrophoresis. As can be seen in Fig. 3. HLE isozymes stain for carbohydrate with tive than cu-chymotrypsin against N-CBZdifferent intensities. Assuming that the I>-tyrosine-p-nitrophenyl ester. Therefore. contamination with the known, chymo- relative intensit!, of’ staining reflects the RESULTS

TABLE

Total mg Protein” Patient CR Crude lysosomal Purified HLE” Patient CD Crude lysosomal Purified HLE

I

Total activity NRA esterase units

Specific activit>units/mg

Fold purification

extract

3.17 0.50

9%1 Yci74

“918 15.11x

1 5.2

extract

7.38 0.84

11.122 9927

l.i.18 11.818

1 7.6

” Following affinity chromatography and (NH,),SO, ’ From the lysosomes of l-2 Y 10y leukocytes.

concentration.

HUMAX

LEUKOCYTE

ELASTASI?

95

extracts cochromatograph with a K,, value to a molecular weight of corresponding approximately 22,000. A similar value is obtained by dodecyl sulfate-gel electrophoresis. but only after certain precautions are taken in handling the samples. If’ HLE is prepared for electrophoresis by heating at 37°C for 2 h in 1”; SDS and 15 Z-mercaptoethanol. extensive autolysis of’ the protein occurs. Three protein bands appear: the two larger ones have apparent molecular weights of 12.000 and 10.000. In addition, there is a hand which migrates with the tracking dye (Fig. 5a). Only the larger peptides have PAS-staining material. The autolvtic products seen in Fig. 5a occur only in the presence of’ dodecyl sulfate and 2-mercaptoethanol. No difference in do-

FIG. 1. Polyacrylamide gel electrophoresis of purified HLK at pH 4.5. Right, active H1.E: center. PMSF 13.4 mM)-inactivated HI,E: and left. mixture of active and inactive preparations. Direction of mtgration was toward the cathode (hottom).

total carbohydrate content. it would appear from Fig. 3 that the slower migrating isozymes have increased carhohvdrate to amino acid ratios. This was confirmed by densitometry of the gels. quantitating the area under each isozyme peak. The results (Table II) suggest that the slower migrating isozymes have carbohydrate to protein ratios 2.4~1.6 times greater than the fastest migrating band. The nature of’ this carbohydrate has not been investigated. Electrophoretic mobility of’ leukocyte elastase. however, is unaffected by treatment with neuraminidase, though the mobility of cu,AT is considerably altered by treatment with neuraminidase under identical conditions. The molecular weight of’ HLE preparations has been determined by Sephadex G-75 column chromatography and dodecyl sulfate-acrylamide gel electrophoresis. Results of’ the Sephadex experiments are found in Fig. 4. HLE elutes a little behind chymotrypsinogen A. NRA esterase activity and protein of’ purified HLE and the NHA esterase activity in crude lysosomal

FIG. 2. Hydrolysis of elastin by purified HLE isozymes following polyacrylamide gel electrophoresis at pH 4.5. The first three gels from the left were photcqraphed in a dark field. Dark areas, therefore, represent elastin digestion hy HLE isozymes. Increasing concentrations of HLE were subjected to electrophoresis. Gels were then submersed for 5 min in a Tris-M&I, $-alanine solut ton t Materials and Methods) and incubated at :37”C for 6 h. The gel on the right was photographed in a light field and is the Naphthol trlue black stain trt’ the HLE isozymes. Direction of migration is twvard the cathode (hottom).

96

TAYLOR

AND CRAWFORD

/JCL\ \/ ;* * FIG. 3. HLE isozymes following electrophoresis at pH 4.5. Top. Naphthol hlue black stain for protein: and bottom. PAS stain for carbohydrate. Densitometric tracings made l’ollowing complete destaining are shown above each gel. Densitometry of gels for protein was performed at 660 nm and for carhohydrate at 540 nm. Isozgmes are numbered arhitrarily from the fastest (I) to the slowest (III) migrating protein hands. Direction of migration was toward the cathode (right).

decyl sulfate-gel patterns can be detected between freshly isolated HLE and HLE heated at 37°C for 24 h when incubation is performed without dodecyl sulfate and mercaptoethanol. Approximately 22% of the NBA esterase activity of HLE is retained in the presence of I? dodecyl sulfate (Fig. 6). This appears to be the critical factor for autolgsis because porcine pancreatic elastase, which is completely inactivated in 15 dodecyl sulfate (Fig. 6). does not undergo autodigestion (gel not shown). Moreover, PMSFinactivation of the NBA esterase activity of HLE prior to sample preparation completely inhibits autolysis (Fig. 5b). Heating preparations to 100°C for up to 2 h is insufficient by itself to completely inhibit proteolysis (Fig. .ic); therefore, PMSF is added prior to dodecyl sulfate and reducing agents in preparing samples for molecular weight determination. Samples then are

heated to 100°C for 1 h or more to be certain the maximum amount of dodecyl sulfate becomes bound to the protein. Another prerequisite to obtaining consistent values in molecular weight determinations using dodecyl sulfate gels is the addition of 2-mercaptoethanol during sample preparation. When reducing agent is omitted during heating, regardless of whether PMSF is present or absent, artifacts appear. Heating preparations to 100°C for 4 min under these conditions results in three protein bands following electrophoresis (Fig. 5d). The molecular weights of these bands have been found to be quite variable, ranging from 22,000 to TABLE RELA.TI\E

Bands*

II

CAHHOIIYDHATE CONTENT ISOZVMES

Carbohydrate” Protein

OF ELASTASE

Relative amount carbohydrate

” Bands are numhered arhitararily from the fastest migrating protein (I) to the slowest (III). “Total PAS-staining material was determined by calculating the area of each peak scanned at 540 nm and for protein the area scanned at 660 nm. The data represents the ratio of the two values.

FIG. 4. Sephadex G-75 elution profile of HLE (fraction vol 0.42 ml). Circles represent NBA esterase activity of purified HLE (-1 and crude Iysosomal extracts (-----I. Open squares represent protein of purified and PMSF-inactivated HLE. Calibration curve for the column (0.9 Y 22 cm) is included in the insert using ovalhumin. chymotrypsinogen A and ribonuclease A as standards. The arrow indicates the K,, value for HLE.

HUMAN

LEUKOCYTE

Interaction

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified HLE. (al HLE incuhated at 37°C (2 h) prior to electrophoresis as described in Materials and Methods; (h) same conditions as (a) except that PMSF (3.4 IIIM) was used prior to sample preparation to inactivate the enzyme; (cl HLE heated to 100°C for 2 h prior to electrophoresis without PMSF; (d) HLE heated to 100°C for 4 min prior to electrophoresis in the absence of 2-mercaptoethanol. In order to clearly separate the three polypeptides in (a) electrophoresis of gels was begun at :i mA/tuhe, increased to 4 mA/tuhe after the protein had entered the stacking gel and to 5 mA/tuhe after entering the separating gel.

97

ELASTASE

of HLE with cy,AT

Preliminary studies were undertaken to examine the interaction of HLE and (u,AT. Different amounts of HLE (O-52 pg) were mixed with 23 pg cr,AT, then PMSF (final concentration 3 mM) in a volume of 70-80 ~1 at room temperature. Both HLE and (u,AT were in 0.025 M sodium pyrophosphate buffer, pH 5.9, with lOc/csucrose and 0.035 M /3-alanine. Following mixing, the samples were prepared for dodecyl sulfate-acrylamide electrophoresis at 100°C as described in Materials and Methods. As seen in Fig. 7, LU,AT forms one major complex with HLE at low concentrations. The molecular weight of N,AT (using pepsin, bovine serum albumin, and chymotrypsinogen A as standards) is 45,000 under these conditions. The protease-inhibitor complex is 6’7.000 daltons, a molecular weight predicted if HLE were to complex with (Y,AT at a 1:l molar ratio. With increasing concentrations of HLE, however, the larger molecular weight complex decreases in staining intensity as an intermediate form of M, 60,000 appears (Fig. 7). The complexes formed under these conditions have been observed to be stable to electrophoresis at pH 4.5 (data not shown) in addition to dodecyl sulfateeacrylamide gel electrophoresis, indicating the formation of a “pseudocovalent” bond between protease and inhibitor. Inactivation of HLE with PMSF prior to addition ofcu,AT. however, completely inhibits the formation

39,000. Heating samples in 1%. dodecyl sulfate and 1% 2-mercaptoethanol at 100°C for 1 h (with PMSF) results in a major band at it!, 22,000 with a minor “tail” of protein extending to M, 24,000 (similar to that seen in Fig. 5b).

K, and V Kinetic parameters of HLE were examined using the synthetic substrate NBA. At 25°C and pH 8.3 (0.025 M sodium pyrophosphate) the K, was found to be 0.36 mM and V to be 39 nmol p-nitrophenoll min.

FIG. 6. The effect of dodecyl sulfate on the NBA esterase activity of porcine pancreatic elastase (closed circles) and HLE (open circles). Dodecgl sulfate was mixed with enzyme preparations, which were then added to assay buffer and monitored for release of p-nitrophenol as described in Materials and Methods.

98

TAYLOR

b

AND CRAWFORD

c

FIG. ‘i. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mixtures of n,AT and HLE. (a) 23 ~g cr,AT, (b) 23 /.~cga,AT + 6 /.q HLE. (cl 2.3 wg u,AT + 19pgHLE. and (d) 23pga,AT t 52figHLE.

of stable complexes visualized by dodecyl sulfate-gels or by electrophoresis at pH 4.5 (data not shown). This further suggests that the “pseudocovalent” bond formed between inhibitor and enzyme requires that the protease active site be intact. DISCUSSION

We describe a method for isolating HLE in good yield from the granules of the PMS leukocytes obtained from 500 ml of blood. Approximately 1.5-3.0 mg of purified enzyme can be obtained in 2-3 h after lysis of’ the granules. Although this is not a large amount of protein, modern methods allow appreciable structural information to be deduced from this quantity of material. A distinct advantage of the method is that it also succeeds with PMSF-inactivated HLE. In view of the tendency of active enzyme to undergo autolysis under certain conditions, inactivated enzyme should be valuable in the further analysis of the enzyme’s structure. Purified HLE appears to consist of a single polypeptide chain with a molecular weight near 22,000. It forms a complex pattern of isozymes following electrophoresis in acid acrylamide gels (Fig. 1). Preliminary evidence suggests that the electrophoretic heterogeneity observed results

from different amounts of carbohydrate attached to the molecules (Fig. 3 and Table II). Neuraminidase treatment of HLE for 24 h, however, does not alter the electrophoretic mobility of any of the isozymes, suggesting that sialic acid, if present, is not susceptible to cleavage by the enzyme. Kesistance to neuraminidase digestion is known to occur when sialic acid is sub stituted at the 4-hydroxyl group or linked to a sugar partner which is substituted with other sugar residues (27). The first type of neuraminidase resistance occurs in glycoproteins and equine submaxillary erythrocyte glycosphingolipids, while the second type is found brain monosialoganglioside. A careful analysis of carbohydrate components of HLE should reveal the chemical nature of the isozymic differences. In contrast to our observations, Ohlsson and Olsson (11) have described the purification of leukocyte elastase isozymes with molecular weights of :322:16.000 on dodecyl sulfate gels. Rindler-Ludwig et a/. (X3), using G-75 column chromatography. also obtained a molecular weight of 33,000 for elastase-like proteins contained in human neutrophil granules. The source of material may account for the differences in the molecular weights obtained. Lysosomal granules of PMN leukocytes from patients with untreated chronic myeloid leukemia were used as source material by these researchers while granules from normal individuals were used in the present study. Schmidt and Havemann (12) using leukocyte granules from healthy human donors obtained a molecular weight for HLE of 2’7,006 by gel filtration. Proteolysis of HLE during purification does not appear to be the cause of the even lower molecular weight obtained in the present study. Crude lysosomal extracts treated with PMSF during extraction and/or prior to Sepharose-elastin chromatography yielded a molecular weight for purified, inactive HLE identical to purified active material. Moreover, the NHA esterase activity of crude lysosomal extracts coelutes from Sephadex G-75 with inactivated and purified HLE protein (Fig. 4). Therefore. if proteolysis were responsible for the lower

99

HUMANLEUKOCYTEELASTASE molecular weight we observed, it would have to occur during extraction and be catalyzed by an enzyme which is not affected by PMSF. This seems unlikely because the extraction methods used by others (11, 12. 28) are similar to the one we have used. One problem we have noted in using gel filtration to determine the molecular weight of HLE in crude lysosomal extracts concerns proper selection of pro tein and/or salt concentration prior to addition to the column. Crude lysosomal extracts (in 1 M NaCl) are cloudy when protein is at a concentration of approximately 4 mg/ml. HLE in this preparation eluted slightly ahead of chymotrypsinogen A. When the same preparation was diluted with pyrophosphate buffer without NaCl, the turbidity disappeared. The HLE then eluted behind chymotrypsinogen A with a K,,. value corresponding to a molecular weight of 22,000 (Fig. 4). If dodecyl sulfate acrylamide gel electrophoresis is used, other factors may be responsible for the varied molecular weights obtained. Glycoproteins have been shown to be impaired in dodecyl sulfate binding (29-31). which may yield drastically lowered electrophoretic mobilities (30, 32). The increased amounts of carbohydrate in slower migrating isozymes (Fig. 3) could contribute to incomplete dodecyl sulfate binding. The ultimate effect in dodecyl sulfate gels would be slower electrophoretic mobilities and larger molecular weights. This may account for the artifact seen in Fig. Fid. At the onset of this study we obtained apparent molecular weights similar to those reported previously (11. 12, 28); however. by varying the methods of sample preparation. lower values were consistently obtained. Through prolonged heating (in the presence of mercaptoethano1 and PMSF) a major protein band near M, 22,000 is invariably found. A trailing “smear” of protein is always seen in these preparations extending to a molecular weight of approximately 24,000 which may represent the minor isozymes with increased amounts of carbohydrate and slightly larger molecular weights. The NBA esterase and elastolytic activity of HLE was unimpaired by exposure

to 8 M urea. Moreover, 225 of the NBA esterase activity remains in 0.1-l%’ dodecyl sulfate at 22°C. The latter observation suggests that a limited amount of dodecyl sulfate becomes bound to the molecule. distorting but not completely disorganizing the active site region. Intramolecular disulfide bridging. as found in the pancreatic serine proteases, probably contributes to the unusual stability of the molecule. Autolysis in 1“; dodecyl sulfate, as monitored by dodecyl sulfate-acrylamide gel pat terns, becomes noticeable only in the presence of mercaptoethanol concentrations high enough to reduce disulfide bonds (Fig. 5). It is interesting to note that subtilisin. a serine protease in a family different from pancreatic trypsin and elastase. also undergoes autolysis under similar conditions (33) as does a serine protease of the fungus Malbranchea pulchella (34). In an attempt to ascertain the importance of disulfide bridging to the integrity of the molecule in the absence of denaturants we performed acrylamide gel disc electrophoresis at pH 4.5 on a sample of purified HLE reduced with 20% P-mercaptoethanol. The isozymic pattern and mobility were unchanged from normal by this treatment (unpublished observations). Interaction

of CY,AT with

HLE

Incubation of cu,AT with low concentrations of active HLE results in the formation of a stable complex with a molecular weight of 67,000. As the concentration of HLE is increased. a new complex of M, 60.000 appears. This is probably the result of proteolysis of o(,AT by HLE, possibly in a manner similar to that which occurs with trypsinLu,AT complexes (35). In our experiments no inhibitor-enzyme complexes larger than M, 67,000 were observed, even in protease excess. This leads us to conclude that n,AT reacts with HLE in a 1:l molar ratio. The inability of a,AT to bind to PMSFinactivated HLE was at first surprising. In 1953. however. Green (36) noted that disopropylfluorophosphate-inactivated trypsin does not form complexes with ovomucoid, soybean trypsin inhibitor, or pancreatic trypsin inhibitor. This eventually led to the

100

TAYLOR

AND CRAWFORD

elegant work of Finkenstadt and Laskowski (37) and Ozawa and Laskowski (38) in which they proposed a general mechanism of trypsin inhibition. The mechanism involves the hydrolysis of a specific peptide bond in the inhibitor and the formation of an ester bond between the active serine of trypsin and the newly created free carboxyl group of the inhibitor. This would suggest that there are areas on the polypeptide chain of the inhibitor which are specifically recognized by the protease. Such a mechanism could explain the differences in the ability of (u,AT to combine with and inhibit a variety of proteolytic enzymes by assuming that cu,AT does not contain specific substrate recognition sites for those proteases which are poorly inhibited. Certainly much work remains to be done in regard to the elucidation of the mechanism of cu,AT-HLE interactions. The similarity, however, between our observation and that made by Green over 20 yr ago is unmistakable and strongly suggests that the mechanism of cu,AT inhibition of proteases may be the same as that proposed for wellknown trypsin inhibitors (37. 38) or for other inhibitors of serine proteases such as the anti-thrombin-heparin cofactor (39). ACKNOWLEDGMENT We wish to thank Daniel Twumasi for communicating to us the potential of Sepharose-elastin as a purification technique for leukocyte elastase. REFERENCES 1. ERIKSSO~, S. (196.5) Acta Med. Stand. 177, Suppl. 432, 6-85. 2. MASS, B., IKEDA, T., MERANZE, D., WEINBAUM, G., AND KIMBEI., P. (1972) Amer. Rec. Resp. Dis. 106, 384-391. 3. KAPLAN, P. D., KUHN, C., AND PIERCE, J. A. (1973) J. Lab. C/in. Med. 82, 349-356. 4. WEINBAI.M, G., MARTO, V., IKEDA, T., MASS, B.. MERANZE. D. R., AXD KIMBEI., P. (1974) Amer. Reu. Resp. Dis. 109, 351 -357. 5. SNIDER, G. L., HAYES, J. A.. FRANZBLAI, C., KAGAN, H. M., STONE, P. S.. AND KORTH~, A. L. (1974) Amer. Rec. Resp. Dis. 110, 254-262. 6. LAZARUS, G. S.. DANIELS;, J. R., BROWN, R. S.. BLADE%, H. A., AND FULLMEH, H. M. (1968) J. Clin. Inuest. 47, 2622-2629. 7. JANOFF. A., ANI) SCHF.KEK,J. (1968) J. Erp. Med. 128, 1137-1151. 8. BARNHAHT, M. I.. QUINTA~A, C.. LENON, H. L..

9. 10. 11. 12. 13.

14. 15. 16. 17.

18. 19.

20. 21. 22. 23. 24. 25. 26.

27.

28.

29. 30. 31. 32. 39.

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ELASTASE

101

37. FINKENSTADT, W. R., AND LASKOWSKI. M.. JR. (1965) J. Biol. Chem. 240, PC 962-PC963. 38. OZAWA. K., AND LASKOWSKI. M., JR. (1966) J. Bid. Chem. 241, 3955-4961. 39. HIoHSMITH, R. F., AND ROSE:NBEH(:.R. D. (1974) J. Sol. Chem. 249.4335-43338.