Norman M. SchechterS, Linda M. Jordan, and April M. James. From the Departments of Dermatology and Biochemistry and Biophysics, School of Medicine, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 31, Issue of November 5, pp. 2362623633,1993 Printed in U.S.A.
Reaction of Human Chymase with ReactiveSite Variants of cul-Antichymotrypsin MODULATION OF INHIBITOR VERSUS SUBSTRATEPROPERTIES* (Received for publication, March 11, 1993, and in revised form, July 9, 1993)
Norman M. SchechterS, Linda M. Jordan, andApril M. James From the Departments of Dermatology and Biochemistry and Biophysics, Schoolof Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Barry S . Cooperman From the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Zhi mei Wangand Harvey Rubin From the Departments of Medicine and Microbiology, Schoolof Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 ~~
Inhibition of human chymase by al-antichymotrypHuman chymase from skin is a chymotrypsin-like proteinsin produces 3.5 mol of degraded inhibitor for every ase that is stored within the secretory granules of mast cells mol of chymase inhibited, resulting in a stoichiometry (1-3). We have shown previously that this proteinase is inof inhibition (SI) of 4.5. In the present study, thesub- hibited by two human plasma inhibitors, ACT’ and al-prostrate versus inhibitor propertiesof this reaction were teinaseinhibitor (4). Bothinhibitorsare members of the examined further using wild type and mutant recom- serpin(serineproteinaseinhibitor)protein family (5, 6). binant antichymotrypsins (rACT). Titration of chy- Inhibition of chymase by both inhibitors appeared irreversimase hydrolytic activity with rACT-L358(wild type) ble, showing the typical tight binding SDS-stable complexes (Pl) variants of ACT, L358W, characteristic of serpin-target proteinase interactions (5, 6). andreactivesite L368M, and L358F revealed that the SI was sensitive Apparent second-order rate constantsobtained under pseudoto P1 residue replacements. SI values increased in the s” for ACT and 7,500 order of Trp < Met c Leu c Phe whereSI values were first-order conditions were 21,000 1.5, 2, 4, and 7 , respectively. Chymase inhibitor com- M” s-’ for al-proteinase inhibitor (4). The magnitude of plex and cleaved inhibitor were demonstrated by so- these values is on the low side for serpin-proteinase interacdium dodecyl sulfate-polyacrylamide gel electropho- tions, approximately 1,000 x lower than values reported for resis for all variants; the relative intensities of each the inhibition of neutrophil cathepsin Gby ACT and neutroband were consistent with SI values established by phil elastase by al-proteinase inhibitor (7). The reaction of titration. NHa-terminalsequence analyses of the prod- chymase with ACT and al-proteinase inhibitoralso displayed ucts formed in the reaction of chymase with rACT- an unusually high SI, a value empirically defined as mol of L358F indicated that thePI-PI‘ bond was the primary inhibitor required to inhibit 1 mol of chymase measured by site of cleavage resulting in the hydrolysis and inacti- titration. SI values of 4.5 and 5.0 were obtained for inhibition vation of this variant. The apparent second-order rate of chymase by ACTand al-proteinaseinhibitor, respectively. constant for chymase inhibition (k’/[I]) by rACT also Further analysis of the interaction of chymase with each was affected by P1 substitution. k’/[I] values increased inhibitor revealed that the high SI for inhibition was caused in an orderopposite that obtained forSI values (Phe < by a competing reaction producing a hydrolyzed inactive Leu < Met < Trp). The reactive loop mutant (rACT- inhibitor. SDS-PAGE and NH2-terminal sequence analyses P3P3’) produced by replacing the reactive site region indicated that the hydrolyzed inhibitor was generated by of ACT (Thr36e-Va13e1) with that of al-proteinase in- cleavage within the reactive loop (4); the cleavage site for hibitor (Ile36e-Prose1 ) revealed a different reaction patACTwas provisionally located at the reactive site of the tern. Although its SI was near 1, the value for k’/[I] inhibitor ( L e ~ ~ ~ ~ - S ewhereas r ~ ~ ’ ) ,the cleavage site for a1was the lowest among variants. rACT-L358R, another proteinaseinhibitor was located at a bond (Phe352-Le~353) P1 variant, did not inhibit chymase. These results are several residues away from the reactive site (Met358).SI values evaluated with respect to the substratepreferences of obtained from titrations of chymase with either ACT or a1human chymase andwith respect topartitioning schemes proposed to explain SI values greater than 1. proteinase inhibitor did not vary as a function of the initial enzyme concentration, indicating that the fraction of total inhibitor converted to hydrolyzed inhibitor was an invariant property of each reaction (4). In this work, we provide additional evidence that the loca* This work was supported by National Institutes of Health Grant AR39674 and by a grant from H & Q Life Sciences, San Francisco. tion of the chymase cleavage site producing hydrolyzed ACT
“’
The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Dept. of Dermatology, Clinical Research Bldg., University of Pennsylvania, 422 Curie Blvd., Philadelphia, PA 19104.Tel.: 215-898-3680;Fax: 215-573-2033.
The abbreviations used are: ACT, cyl-antichymotrypsin; SI, stoichiometry of inhibition; PAGE, polyacrylamide gel electrophoresis; rACT, recombinant antichymotrypsin; pNA, p-nitroanilide; HPLC, high performance liquid chromatography; PVDF, polyvinylidene difluoride; MOPS, 4-morpholinepropanesulfonicacid SUC,succinyyl; PMSF, phenylmethysulfonyl fluoride.
23626
Reaction of Human Chymase Variant withAntichymotrypsins is the reactive site of the inhibitor whichis referred to asthe P1-P1' site following the nomenclatureof Schechter and Berger (8). We also investigate further the substrate uersus inhibitor properties of the reaction of chymase with ACT using recombinant ACT (rACT) variants. These studies provide additional support forthe inclusion of a rapid partitioning schemein the generalequationfor serpin-proteinase interactions to explain high SI values, as proposed by Cooperman et al. (9) in the preceding article. EXPERIMENTALPROCEDURES
Materials-Peptide-pNA substrates were purchased from Sigma or Bachem. Bovine chymotrypsin and trypsin were from Calbiochem or Sigma. TSK-heparin-5PW and Mono Q HPLC columns were from Supelco and Pharmacia LBK Biotechnology, Inc., respectively. Immobilon-P membranes (PVDF) were from Millipore. Heparin-Sepharose was from Pharmacia. The detergents dodecyl maltoside and Triton X-100 were obtained from Anatrace and Sigma, respectively. rACT Variants-Expression of rACTs, production of reactive site, reactive loop (rACT-P3P3'), and cassette mutants of ACT, and purification of rACTs, were accomplished as described by Rubin et al. (10) and Kilpatrick et al. (11). rACT-Leu35&as, rACT-L358Fcas, and rACT-PBP3'cas refer to variants produced using acassette construction (11).Insertion of the cassette into ACT cDNA required the creation of two restriction sites which resulted in three amino acid changes in the inhibitor. In one, Ala3''-Alaw (PlO-Pg) was changed to Gly-Thr, and in the other, Valrn (P10') was changed to Thr. Sites P9-P10 are located near reactive loop position P14, a site critical for serpin inhibitory function (12-15). Mutations at site P10 of antithrombin I11 (Alam to Ser or Pro) and C1 inhibitor (Ala"' to Thr) have been shown to reduce the inhibitory activities of these serpins (13). The P10 Ala to Gly mutation in our cassette variantsof ACT was a more conservative substitution than those affecting the activity of antithrombin 111. In addition,the P10 site of cY1-proteinase inhibitor is Gly, suggesting that this amino acid residue maybe acceptable in the P10 position of ACT. These changes alone did not alter the inhibition properties of ACT toward chymotrypsin (11). rACTs also contained a short NH2-terminal extension of 8 (10) or 4 (in more recent constructions) aminoacids. Inhibition of chymase by rACT-Leu3" variants containing both types of NHz-terminal constructions was identical with respect to SI and inhibition rate constants. Purification of Human Chyme-Mast cell proteinases were extracted from skin and fractionated on a 500-ml heparin-Sepharose column (1, 16, 17). Chymase was eluted from the column in a single step with a solution of 2 M NaCl, 0.1 M MOPS (pH 6.8), and was further purified by affinity chromatography using soybean trypsin inhibitor-Sepharose (1, 16, 18). This material was used in most studies. For reasons still unclear, the fraction of chymase in skin extracts which binds to heparin-Sepharose is variable (18).In extracts in which 30-70% of the enzyme did not bind, an alternative purification method was used. Chymase not bound to heparin-Sepharose was precipitated by the addition of protamine chloride (1, 18).After solubilization of chymase in high salt buffer, the preparation was fractionatedon phenylbutylamine-Sepharose.Bound enzyme was eluted with a solution of 0.4 M NaCl, 0.01 M MOPS (pH 6.8), 0.2 M D-Trp-OMe. Chymase was then chromatographed on a50-ml heparinSepharose column. The proteinase now bound to thecolumn and was eluted in high salt buffer as described above. As far as we can detect, chymase purified by both procedures has identical catalytic, physical, and inhibition properties; therefore, distinctions between the two chymase preparations were not made in the text. TSK-Heparin Chromatography-Additional fractionation of chymase was achieved on a TSK-heparin column eluted with an increasing NaCl concentration gradient. Chromatography was performed using a Perkin-Elmer-Cetus Instruments series 410 HPLC pump and series 235 diode array detector. Chymase isolated as described above was loaded onto the column in a solution of 0.4 M NaC1, 0.01 M MOPS (pH6.8) and theneluted with a linear NaCl gradient ranging from 0.4 to 1.6 M NaCl. Fractions were assayed for chymotrypsin activity understandard conditions to obtain initialrates of hydrolysis, or they were allowed to incubate in substrate solutions for upto 24 h to detect minor contaminating proteinases. In the latter assays, 20pl samples from column fractions were incubated in 250 pl of assay buffer containing 2 mM Suc-A-A-P-F-pNA. Reactions were stopped
23627
by the addition of0.5 ml of 5% acetic acid and were quantified spectrophotometrically. Determination of Inhibitor and Proteinase Concentrations-The concentrations of rACTs were determined by titration of a standard amount of chymotrypsin, except for rACT-L358R, which was quantified using trypsin insteadof chymotrypsin. Titrations were routinely performed by the addition of varying amounts of inhibitor to 50-100p1 reactions containing approximately 200 nM proteinase, 0.1 M TrisHCl (pH 8.0), 0.5 M NaCl, 0.01% dodecylmaltoside (25 "C). Residual activity was measured spectrophotometrically after 10-15-min incubations by dilution of an aliquot of the reaction mixture into 1 ml of assay buffer containing 1 mM Suc-A-A-P-F-pNA or benzyloxycarbonyl-GPA-pNA. Stock chymotrypsin and trypsin solutions were standardized with the active site titrantsN-trans-cinnamoylimidazole (19) or p-nitrophenylp'- guanidinonitrobenzoate (20). Chymase concentrations were determined under standard assay conditions (0.4 M Tris-HC1 (pH 8.0), 1.8 M NaCl, 9% MezSO, 1mM Suc-A-A-P-F-pNA) assuming a specific activity of2.7 pmol of product min"/nmol of chymase p-nitroaniline = 8,800 M-' cm"). This valuewas determined previously using titration with lima bean trypsin inhibitor to obtain the concentration of chymase (4). Titrations and Time Course Studies-Reactions of chymase with rACTs were performed at 25 "C in 50-150 p1 of a solution containing 0.2 M Tris-HC1 (pH 8.0), 1 or 2 M NaCl, and 0.01% Triton x-100. After incubation with various amounts of inhibitor for appropriate times, residual activities were measured spectrophotometrically after dilution (25-50-fold) of a sample aliquot into 1 ml of standard assay buffer containing 1 mM substrate. Rates of substrate hydrolysis were constant over the 3-min period used to determine residual activities, indicating that chymase-ACT complexes were stable to dilution. Inhibition Rate Constants-Progress curves were determined under pseudo-first-order conditions ([I10 >> [E],) in the presence of substrate as described by Petersen and Clemmensen (21). Reactions contained 0.25-1.0 mM Suc-A-A-P-F-pNA,2.5-3.0 nM chymase, and inhibitor. Absorbance was monitored continually for 15 min, and instantaneous velocities were determined over every 1-min interval by linear regression analysis of the data. Plots of the instantaneous velocity uersus time ( t ) were fit by nonlinear methods to the expression V0e-U to determine hob, the observed first-order rate constant in the presence of substrate; Vo corresponds to initial activity. The apparent second-order rate constant determined after correcting for the presence of substrate is equal to k'/[I], where k' = k,(([S]/K,) + l ) , [I] is the inhibitor concentration, [SI is the substrate concentration, and K , is the Michaelis constant for chymase hydrolysis of the substrate under the experimental conditions. Reactions were monitored for at least three half-lives, except for rACT-P3P3', where data covered one half-life. K, for hydrolysis of substrate by chymase was 0.80 mM in 1.0 M NaCl and 0.49 mM in 2.0 M NaCl (4). SDS-Polyocrylnmide Gel Electrophoresis-Reactions of chymase with inhibitors under standard titration conditions were stopped at appropriate timesby the addition of PMSF. After a 15-min incubation a t 25 "C in 0.4 mM PMSF, samples were denatured in SDSby heating at 90"C for 10 min. Products were resolved onSDS gels (10% acrylamide, 0.33% N,N"methylene bisacrylamide) under reducing conditions and visualized by staining with Coomassie Brilliant Blue. NH2-terminal Sequence Analysis-The variant evaluated was rACT-L358Fcas because it had the highest SI. Reaction conditions were similar to titrationsexcept that the concentrations of inhibitor and enzyme were significantly higher. NHZ-terminalsequence analyses wereperformed on either complete reaction mixtures or reaction products electroblotted onto a PVDF membrane (22). In the latter experiments, reactions were performed at an [IlO of 2.5 p~ and an [I] o/[E]O molar ratio of 10, a value slightly greater than theSI. The high [I10 and SI should minimize the possibility of nonspecific proteolysis of ACT by chymase. After a 30-min incubation at 25 OC reaction mixtures containing630-pmol equivalents of rACT were concentrated by lyophilization, and salt was removedfrom the resulting precipitate by repeated washing in cold 80% EtOH. The precipitate was then dissolved in 2% SDS, heat denatured, resolved on a highly crosslinked SDS gel (17.5% acrylamide, 0.58% N,N"methylene bisacrylamide), and electroblotted onto a PVDF membrane. The membrane was stained with Coomassie Brilliant Blue, and the area containing a broad band centered at a M , of 4,000 was cut from the membrane and subjected to automated Edman degradation. In experiments in which whole reaction mixtures were subjected to automated Edman degradation, incubations of chymase with rACT-L358Fcas were performed at 4 "C for 20 min. [I]o/[E]o ratios were 15 and 26, and [IlO was 1.7 and 5.6 pM, respectively. PMSF was added to the reaction
23628
Reaction of Human Chymase with Variant Antichymotrypsins
mixtures at the conclusion of the incubation period. After a further 20-min incubation, samples were concentrated, desalted as described above, dried under nitrogen, and finally resuspended in trifluoroacetic acid. A portion of the trifluoroacetic acid-solubilized sample was analyzed by automated Edmandegradation, and another portion was subjected to quantitative amino acid analysis to confirm the protein concentration. rACT-L358Fcas reacted with chymase in the latter analyses was subjected to fractionation on a Mono Q column as an additional purification step. Kinetics Constants for Peptide pNA Substrates-K, and k, values for the hydrolysis of Suc-A-A-P-F-pNA by chymase insolutions containing 1 and 2 M NaCl were determined according to the Michaelis-Menten rate equation by nonlinear regression analysis of Six to eight initial velocity versus substrateconcentrationdata. different substrate concentrations ranging from 0.25 to 3 mM were used in each experiment. Fits were all statistically significant with standard errors in K,,, and katvalues of < 18 and lo%, respectively.
and by rACTs are an intrinsic property of the interaction between these proteins. Reaction of Chymase with Variant rACTs-Titrations for five reactive site (P1 site) variants denoted as rACTs L358 (wild type), L358M, L358F, L358W, and L358R, and a more extensively substituted variant, denoted as rACT-P3P3’, are shown in Fig. 2. In the latter variant, the P3-P3‘ sequence of ACTwas replaced by the corresponding sequence of crlproteinase inhibitor. The difference in the titrations demonstrate that the SI value was sensitive to mutation of the P1 site aswell as to mutation of the P3-P3’sequence. The variant rACT-L358R did not inhibit chymase even at an [I]o/[E]o ratio of 20 (not shown in Fig. 2). SI values for each variant are reported in Table 1 along with SI values for three additional rACT variants that were made using a cassette construction (denoted as cas) as described under “Experimental RESULTS Procedures.” Examples of time courses for reactions show The Large SIfor theReaction of Human Chymase withACT that the inhibition obtained at different [I]o/[E]o ratios was Is Not the Result of a Contaminating Proteinase or Poorly stable, even at the high ionic strength (1M NaC1) conditions Folded Inhibitor-To confirm the purity of chymase prepa- employed (Fig. 3). Residual activities in these studies were rations routinely used in reactions with rACTs, an enzyme achieved within minutes and remained constant for at least preparation was subjected to additional fractionation on a 24 h. Consistent with the stability of residual activities, the TSK-heparin HPLCcolumn. Only one peak of chymotrypsin- SDS gel band representing the chymase-rACT complex relike activity was observed eluting from the column a t 0.75 M mained intact for at least 24 h of incubation (Fig. 1B). NaC1, even when fractions were assayed for long time periods Apparent second-order rate constants (k’/[I]) for the inhito allow for minor contaminating activities to be detected. bition of chymase by rACT variants are reported in Table I. The SIvalue for rACT and rACT-L358F inhibition of highly They were measured at [rACT]/[chymaseIo ratios at least 10 purified chymase was the same as thatobtained with routinely times the SI determined for each variant. At these high [I],/ purified chymase, indicating that our routine preparations [E],ratios, we have shown previously that the reaction of were not contaminated with another proteinase capable of chymase with serum ACT follows pseudo-first-order kinetics hydrolyzing rACTs. (4). Chymase activity loss in the presence of all rACT variants In a second test of enzyme purity, chymase purified by was first-order, proceeding to a complete loss of catalytic routine procedures was reacted with rACT at [rACT],/[chy- activity. Inhibitor concentration for these measurements did mase], > SI, and thenreactions were analyzed by SDS-PAGE not exceed 300nM. This concentrationis not likely saturating to show that excess inhibitor was not subject to further for the reaction based on our previous study with serum ACT, hydrolysis by a possible contaminating proteinase. The pres- which showed a linear relationship between k’ and [I] up to ence of intact inhibitor in addition to degraded inhibitor at an inhibitor concentration of 1phi. The results in Table I show that k’/[I] values were also [I]o/[E]o ratios > SI is shown in Fig. L4. Even after long incubation times intact inhibitor was observed on gels (Fig. sensitive to P1 and reactive loop substitutions. k’/[I] values 1B). Residual intact inhibitor was shown to be active in a for P1 variants decreased in the order of Trp > Met > Leu > parallel experiment in which chymotrypsin was added to the Phe, which was opposite to that observed for SI values. This reaction after inhibition of chymase. SDS-PAGE analysis of trend was not followed by the rACT-P3P3’ variant which this reaction mixture (not presented) showed the disappear- demonstrated a low SI and a low k’[I] value.Only minor ance of the intact rACT band and the appearance of an changes in SI and k’/[I] values were observed at the two additional high M , band corresponding to thechymotrypsin- different NaCl concentrations (1 and 2 M ) employed.AlrACT complex. These results also argue against the presence though the reaction properties of cassette variants differed of a proteinase contaminant for which rACT is uniquely a somewhat from those of the corresponding non-cassette variants,the change in reaction properties between cassette substrate. High SI values also could result from the presence of variants paralleled those of corresponding non-cassette variants. Presumably, the different SI andk’/[I] values for both improperly folded inhibitor in our ACT preparations. Previous studies by us have shown that serum-ACT, as well as types of variants are related to the additional mutations at rACT and variantACTs purified from bacterial extracts,have sites P9-PlO and/or P10’ required for construction of the SI values near 1 in reactions with bovine pancreatic chymo- cassette variants. SDS-PAGE Analysis of Products Generated in Reaction of trypsin or trypsin andthat all ACTs behave as single species in experiments that determine their rate constants for inhi- Chymase with rACTs-Banding patterns of reaction products bition of chymotrypsin or trypsin (10, 11).This biochemical obtained for rACTs at indicated [I]o/[E]o ratios areshown in characterization indicates that serum ACT used previously Fig. 4. The patterns clearly show the presence of a complex (4) and the rACTs used in the present study were properly band with an apparent molecular mass corresponding to the folded. The stoichiometries of rACTs used in the present addition of chymase (30 kDa) and rACT (46 kDa), a band workwere determined by titration of each inhibitor with corresponding to rACT when the [IIo/[E]o > SI, as well as a chymotrypsin or trypsin, andonly the concentration of active band or bands migrating slightly slower than rACT (37-40 inhibitor established from these titrations was used to calcu- kDa) presumably corresponding to rACT cleaved in the reactive loop. The relative intensities of these bands vary in late SI values for chymase inhibition. The studies and arguments just described for ruling out qualitative agreement with the SIvalues obtained from titraartifactual possibilities strongly indicate that the large SI tions for each variant; i.e. rACTs with low SI values, like values observed for the inhibition of chymase by serum ACT rACT-L358W and rACT-P3P3’, have an intense complex
Reaction of Human Chymase with Variant Antichymotrypsins
@
Chymase vs rACT - Leu 358 1
2
3
4
5
10"
10"kDa 100 66
-
43
-
kDa
23629
Chymase vs rACT - Leu 358 6
7 --.-
8 .?"
-9
1 0 1 1
100 -
'
4 c
P
4u1
b
4 D I
66 -
4 c
" I 4 UI
43 -
4
30 -
e Chymase
30 21.5
Dl
-
xI = 2.6pM
min
min
min
hr
FIG. 1. Analysis by SDS-PAGE of the products formed in the reaction of human chymase with rACT-L358 as a function of [I]o/[I& ratio (panel A ) and time (panel B ) . Abbreviations on the sides of the panels signify migration of proteinase inhibitor complex ( C ) ,undegraded inhibitor (UI), and degraded inhibitor (DI). The M,of the complex band is roughly the sum of the M, of rACT (46,000) and chymase (30,000). The amount of inhibitor resolved in each analysis was equivalent to 3 pg of native inhibitor; various [I]o/[E]o ratios were obtained by varying [chyma~e]~. Reactions analyzed in panel A were stopped after 30 min by the addition of PMSF. In lanes 2 and 3 there was noticeable degradation of complex bands as well as two bands migrating faster than intact inhibitor. These extra bands are presumably caused by the presence of uninhibited chymase. A single reaction at an [I]o/[E]o ratio of 7 was monitored in panel B. At the indicated times shown below the lanes, reactions were stopped by addition of PMSF (2 mM final concentration) and denaturedin SDS by standard procedures. Denatured sampleswere then analyzed by SDS-PAGE at theend of experiment. Lanes 6 and 7 in panel R are rACT-L358 aloneand chymase alone (1 pg of chymase in this lane does not correspond to that used in the time course). Chymase migrates as broad band because of glycosylation (17). TABLE I Rate constants and SI values for the reaction of human chymase withrACTs In measurements to determine inhibition rate constants, [I] was at least 10-fold higher than theSI determined for that inhibitor with chymase by titration. Apparentsecond-orderinhibition rate constants (k'/[I]) were calculated from the first-order plots as described under "Experimental Procedures." rACT variant NaCl k'l[Il" SI M
MI s-I
1 1.5 151,000 L358W 1 46,500 2.0 L358M 0 2 4 6 0 1 27,000 4.0 L358 l~lJIElo 17.0 24,000 L358F FIG. 2. Titration of human chymase with different rACT 11.3 10,300 P3P3' variants. Chymase concentrations were held constant in each titra2 1.2 280,000 L358W tion, and the amount of inhibitor was varied to obtain the different 2 2.0 64,000 L358M [I]o/[E], ratios. Different symbols denote separate experiments. The 2 42,500 3.5 L358 enzyme concentration used for titration ranged between 150 and 400 2 21,500 7.0 L358F nM. Among individual variants, SIvalues extrapolated from titrations 1 58,000 2.0 L358cas did not change as a function of the initial chymase concentration. L358Fcas 1 12,000 8.0 21.4* 8,250 P3P3'cas band and show little degradation product,whereas those with a Experimental conditions for rate constant measurements were high SI values, like rACT-L358F, have an intensedegradation similar to thoseof titrations except for the presence of substrate and band and show only a small amount of complex. In most 9% Me2S0. Most k'/[I] values are the average of two experiments, analyses, one major band representingdegraded inhibitor was and deviation from the average was typically less than 15%. k'/[I] present; an exception was the reaction of chymase with rACT- values for rACT-L358 in 2 M NaCl and rACT-L258W in 1 M NaCl L358M, where a doublet consistently appeared. Minor bands were based on four and five measurements, respectively; S.D. were 8 and 15% of the reported values, respectively. were observed a t [I]o/[E]o< SI (for example, Fig. 4 A , lanes 4 SI was measured under 1 M NaCl conditions.
and 8; Fig. 4B,lane 2; Fig. 4C, lane 2; and Fig. LA, lanes 2 and 3). The presence of minor bands is presumably due to the further degradation of inhibitor by free chymase. These same reactions also exhibit degradation of the complex band. This degradation did not result in the release of free enzyme, however, asdemonstrated by the time course studiesin Fig. 3. Identification of the Site ofCleavage of rACT-L358F by Human Chyme-SDS-PAGE banding patterns showing major degradation products slightly smaller than rACTs sug-
gest that inhibitors were cleaved by chymase within the reactive loop region (the reactive center is 40 residues from the COOH terminus).Small peptide products (-4 kDa) formed in thereaction of chymase with rACT-L358Fcas were identified on highly cross-linked SDS gels as described previously (4). Following electroblotting of these gels onto PVDF membranes, the small peptides were subjected to NHz-terminal sequence analysis. As shown in Table 11, experiment 1,
23630
Reaction of Human Chymase with Variant rACT-L358 vs Chymase A0.14
I
-
Control
VE = 5.0 -0.02
e
I
0
30
90
60
120
Time, min rACT-L358F cas vs Chymase
-
Bo.10c
Conuol
x
VE = 1.6
a 0.004 0
'
30
90
60
120
Time, min
c
ACT-P3P'3 cas vs Chyrnase
-
0.10
Control
VE = 2.0 e
0.00 I 0
30
60
90
120
Time, min
FIG. 3. Inactivation of chymase by different rACT variants measured as a function of time. Chymase concentrations were 250 nM in panel A, 150nM in panel B, and 150 nM in panel C.
the only peptide identified in the low M , band began with Ser369,the P1' position of ACT. Since rACT-L358Fcas has an SI of 8 (Table I), cleavage arising from its hydrolysis as a substrate should predominate over its hydrolytic product (P1'-COOH-terminal peptide) released upon SDS denaturation of proteinase-inhibitor complexes (23, 24). Thus, only finding peptide beginning with Ser369is a result which indicates thatinhibitor inactivation (hydrolysis as substrate)was produced by cleavage at thereactive site. A similar analysis for the reaction of chymase with serum ACT, which has an SI of 4.5, was described previously (4). Although two apparent sitesof cleavage were observed (Table 11, experiment 4), the peptide corresponding to cleavage at PI-P1' ( L e ~ ~ " - S e r ~was ~ ' )formed apparently in ahigher yield than thepeptide corresponding to cleavage a t P3'-P4' (Leu3'lVal3'*). Since Leu361is located on theCOOH-terminal side of Leu3", we suggest that P3' -P4' cleavage may have occurred following Pl-Pl' cleavage. This suggestion is supported by the results of Baumann et al. (25) who showedthat thepeptide Ser369-Le~36' was removedfrom ACT on prolonged incubation with chymotrypsin. The identity of the cleavage site producing degraded rACTL358Fcas was confirmed in an experiment where NH2-terminal sequence analysis was performed on the entirereaction mixture. The results obtained for two reactions performed at different [I]o/[E]o ratios (ratios = SI and 2SI) demonstrated
Antichymotrypsins
only one NHz-terminal sequence starting at Ser3s9(Table 11, experiments 2 and 3). Based on yields of phenylthiohydantoin derivatives, the estimated recoveries of peptide for the two reactions were roughly 90% and 50%,respectively. Recoveries were estimated assuming that 1) the coupling efficiency of protein to the sequencing support was 50%, and 2) yields of phenylthiohydantoin-Ala andphenylthiohydantoin-Leuin the second and third Edman degradation cycles were representative of peptide recoveries. These high recoveries indicate that themajority of the Ser3"-peptide identified in this analysis was a product generated by the hydrolysis of rACTL358Fcas as a substrate.Amino acid sequences corresponding to theNH2 termini of rACT and chymase were not detected. The absence of the latter sequence was because of its low concentration. The absence of the former sequence is unclear. Although rACT is a bacterial product, other rACT variants prepared for analysis differently have demonstrated a free NH2 terminus. For analyses of whole reaction mixtures, reactions of chymase with rACT-L358Fcas were performed at 4 "C to take advantage of the high SI (SI = 15-17) we observed at this lower temperature. The SI of rACT-L358F, the non-cassette analog of rACT-L358Fcas, also increased as thetemperature was lowered (SI increased from 7 to E ) , and a similar effect of temperaturehas been reported for the reaction of C1 inhibitor will kallikrein (26). SDS-PAGE analyses oflow temperature reactions confirmed the high SI and demonstrated thataltering the temperature hadno effect on the size of the hydrolyzed inhibitor product. The latter observation suggests that the location of the cleavage site producing hydrolyzed inhibitor was not changed by temperature. Changes in SI among Variants Compared to the Substrate Specificity of Human Chymase-Kinetic constants in Table I11 for Suc-V-P-X-pNA substrates were reported by Powers et al. (3), and those for angiotensin I analogs were reported by Kinoshita et al. (27). Human chymase cleaves angiotensin I at the Phes-Hisg peptide bond, thus for this substrate position 8 corresponds to the P1 position (28, 29). As shown in Table 111, the substratepreference (kcatand kCat/Km values) of chymase for a series of P1-substitutedpeptide-pNAsubstrates followed an order (Phe > Leu > Met Trp),which parallels the substrate preference of chymase for rACTs, as shown by the SI values in Table I. Kinetic constantsfor P1-substituted angiotensin I analogs did not follow this order, however. For this series of substrates, the catalytic preference of chymase was Trp Phe > Leu.
-
-
DISCUSSION
Inhibition of proteinases by serpins may occur with SI values greater than 1 as reviewed by Cooperman et al. (9) in the preceding article. The high SI is the result of a concurrent reaction producing a hydrolyzed form of the inhibitor which is inactive. Results presented in this study and a previous study (4) demonstratethat theinteraction of human chymase with ACT is another example of this type of inhibition. The dual outcomes of the chymase-ACT interaction wereevidenced by titrations thatdemonstrated SI values greater than 1 and SDS-PAGE analyses of reactions that demonstrated the presence of botha hydrolyzed inhibitorandastable chymase-ACT complex (Fig. 1). NH2-terminal analysis of the reaction products from inhibitors with high SI values (serum-ACT and rACT-L358F) identified the cleavage site resulting in inhibitor inactivation asthe P1-P1' bond (Table 11). The absence of a minor sequence in theseanalyses indicating asecond proteolytic site within the reactive loop suggests that the P1 site also was
Reaction of Human Chymase with Variant Antichymotrypsins Mr 0
1
2
3
5
4
6
7
23631
9
8
(10”)
-
@
Chymase vs rACT L358F
9666-
10-3Mr
x“
-
43-
2
1
6
7
66 43
-C
-
--uI D ”l-“
-
21.5-
r358AC’
5
4
96 -
kklbna
30-
I/E
3
-
4 Met
a , I
-
4 Phe
@
I I 1
-
8 Met
4, Leu Leu Phe
”
8
2
4
7
6
8
10
-
Chymase vs rACT L358W 1
2
3
4
5
6
10%
4
%-
-C
-
-UI
66
-
43
-
-Dl
30 -
-C
-Chymase UI
Dl
30-
21.5
IIE
2
I I E -
@
66 -
21.5
-
I = 2.5uM
100 -
43
21.5
8
-
13
-
” -
Chymase vs rACT P3P3’ (CAS) 1 0 ” ~
30
-
1
2
4
I = 2.5”
-
I/E I = 2.5pM
- -
12
1.3
3
FIG.4. SDS-PAGE analysis of reactions of human chymase with P1- variants of ACT (panel A ) , rACT-L358F (panel B ) , rACT-P3P‘3cas (panel C),and rACT-L358W (panel D ) . P1 variants analyzed in panel A are reported under each lane; the Phevariant was rACT-L358Fcas. About 3 pg equivalents of (2.5 p~ in reactions) rACT were resolved in each lane. Symbols signifying various reaction products in panels B-D are C, proteinase inhibitor complex; VI, undegraded inhibitor; DI, degraded inhibitor. [I]o/[E]o ratios reported under each lane were obtained by varying [chymaselo. Lunes containing inhibitor alone or chymase alone (paneel D, lane I , contained 1 pg of chymase) are signified by a dash. Reactions (25 “C) were stopped by the addition of PMSF after 1 h (panel A ) or 30 min (panels B-D) of incubation. For reasons that are not clear, degradation products for many variants have slightly different migration patterns. Most notable is the dimeric pattern observed in the case of rACT-L358M (panel A ) and the slower mobility of the rACT-L358Fcas degradation product compared with rACT-L358F (compare major degraded inhibitor product in panel A, lanes 4 and 8, with the corresponding product in panel E ) . Since both Phe variants had approximately the same SI and showed a similar increase in SI when the reaction temperature was lowered, we do not believe that thedifference in the mobility of their hydrolyzed products is indicative of a different hydrolytic site. A t [I]o/[E]oratios < SI there is noticeable demadation of comalex bands as well as the appearance of additional bands migrating below degraded inhibitor bands. I
responsible for chymase inhibition, as expected from inhibition studies with other proteinases (5, 6). This result was different from that obtained for the interaction of chymase with another serpin, a1-proteinase inhibitor, which under certain reaction conditions exhibited an SI of 9 (4).In this case, NH2-terminal analysis demonstrated a minor peptide product corresponding to cleavage at PI-P1’ and a major peptide product corresponding to cleavage a t P6-P7, thereby indicating that the hydrolytic site was different from the reactive site. A second observation supporting the P1 site as the chymase inhibitory site was that replacement of Leu358 with Arg produced an rACT variant active towardtrypsin and chymotrypsin but not chymase. Since human chymase does not readily hydrolyze ester or peptide substrates with Arg a t P1, the lack of inhibition by rACT-L358R agrees with the expectation of reduced interaction at theP1 site. In contrast, chymase inhibition was observed with all other P1 variants of rACT having residues recognized by chymase in substrates.
Other serpin-proteinase pairs that demonstrate reactive sitebond cleavage concurrent with proteinaseinhibitionare thrombin-anthithrombin and kallikrein-C1 inhibitor (26, 31, 32). Analysis of the interaction of chymase with reactive site variants of ACT (Table I) and a more extensively substituted variant, rACT-P3P3’, demonstrated that SI values and apparent second-order inhibition rateconstants (k’/[I]) are altered by mutation. Variationof the P1 residue in rACT with residues recognized by chymase in the P1 site of substrates produced inhibitors with different SI values that followed the order of Trp < Met < Leu < Phe; SI values ranged from 1.5 to 7. k’/[I] values for P1 variants, in contrast, increased following a trend opposite to thatof SI values (Table I). Thus rACT-L358 had the highest inhibition rate constant and the lowest SI among variants. The more extensively altered variant, rACT-P3P3’, which has a reactive site Met, had an SI value near 1 for reaction with chymase. However, its k’/[I]
23632
Reaction of Human Chymasewith VariantAntichymotrypsins
TABLE I1 Zdentificatwn ofchyrnase cleavage sites in rACTs by NH2-terrninalsequence analysis of reaction products Experiments 1 and 4 are of low M, peptide products (Mrbetween 3,000 and 6,000)obtained after resolving reactions on SDS gels and electroblotting proteinsto PVDF membranes. Experiments 2 and 3 are of whole reaction mixtures. The native sequence of ACT from P1’to P15’ is S-A-L-V-E-T-R-T-I-V-R-F-N-R-P.Values in parentheses are netvields reDorted in Dmol. ~~
rACT 1
Exp. 1 L358F (cas) Exp. 2 L358Fb (cas) Exp. 3 L358F (cas) Exp. 4 L358d (a) (b)
~
Cycle
S (5.0)
S (20)
S (22)
3
2
L
A (11.6) (11.5)
5
4
V (7.3)
L
A (117)
(103)
A (153)
(125)
L
V (67)
E (9.4) E (83)
V (79)
I
6
T
R
(3.4)
T (33)
E T (113) (83) (42)
(7.3)
R (60)
R
8
9
T
I
(2.6) (2.3)
10
11
T (2.2)
T
I
T
(31)
(28)
(26)
T
I
T
(39)
(45)
(36)
R (4.4) (4.7)
12
13
F
N (5.6)
R (61)
(80)
F
N (43) (52)
R (112)
F (128)
N (78)
14
R (3.0) R
15
P (2.9) P (49)
A L V E T R T I V R F N R P (18.1) (17) (13.4) (11.7) (4.1) (12.5) (6.1) (4.7) (2.0) (10.5) (5.0) (6.5) (6.0) (3.6) ND’ E T R T I V R F N R P F L M (-4) (3.2) (-4) (1.9) (3.1) (4.0) (-4) (-12) (2.6) (-1.2) (3.6) (7.8) (3.0) (9.4) Thr in position 10 is the result of cassette mutagenesis as described under “Experimental Procedures.” The theoretical yield of product should have been 120 pmol based on the amount of reaction mixture used in the analysis and a resin coupling efficiency of 50%. e The theoretical yield of product should have been 315 pmol based on the same assumptions as described in Footnote b. Two sequences, (a) and (b),were deduced from data. Theresults, but not theactual data, were reported previously (4). e No residue was identified in this cycle because of a high background. Based on the sequence following this cycle it is assumed that serine (a) andvaline (b) are the trueNH2-terrninal residues of these peptides.
ND‘
TABLE I11 Catalytic propertiesof human chymasefor substrates differing inthe identity of the PI residue Kinetic constants for peptide-pNA substrates were determined with human chymase isolated from skin tissue; those for angiotensin I and analogs were determined by Kinoshita et al. (27) with human chymase isolated from heart tissue. Based on the identity of the NHZterminal amino acid sequences (35 residues) for both skin and heart enzymes, they are presumed to be identical proteins (30). Substrate (peptide-pNAor K, aneiotensin I) h u t LIKm mM
SUC-A-A-P-F-NA’ SUC-A-A-P-L-NA SUC-A-A-P-M-NA SUC-V-P-F-NA~
SUC-V-P-L-NA SUC-V-P-M-NA SUC-V-P-W-NA
S-1
M ’s-1
0.8 3.0 2.6
65.0 6.7 2.5
8.1 X 10‘ 2.2 X 103 1.0 X 103
0.1 0.9 1.2 1.6
75.0 21.0 8.7 8.4
7.5 x 2.4 X 7.3 X 5.3 X
lo6 10‘ 103 103
2.1 x lo6 160 0.2 x lo6 46 2.8 X lo6 D-R-V-W-I-H-P-W-H-L 210 ’P1 residues are in boldface. Assay conditions were 0.2 M TrisHCl (pH 8.0), 1.0 M NaCl, 9.0% Me2S0 at 25 “C. Kinetic constants determined in 2 M NaCl (not shown) were similar, except that the Kmvalues were approximately half those obtained in1M NaC1. * Assay conditions were 0.05 M sodium phosphate (pH 8.0), 1.5 M KCI, and 10% Me2S0 at25 ‘C (3). Chymase cleaves angiotensin I at the Phea-His8 peptide bond. Assay conditions were 0.02 M Tris-HC1 (pH 8.0), 0.5 M KC1, 0.01% Triton X-100 (27).
D-R-V-W-I-H-P-F-H-LC D-R-V-W-I-H-0-L-H-L
0.06 0.31 0.11
One involves a branching mechanism described by Cooperman et al. (9) in the preceding article. A compressed version of that scheme sufficient to account for the results presented in this paper is shown below as Scheme 1. Similar branching schemes to this Scheme 1 have been proposed by Bjork et al. (33) and Patston et al. (26). In this model, I binds reversibly to E to form a complex E-I,which is then partitioned along two pathways: one leading to the formation of EI*, a stable enzyme-inhibitor complex, and the otherleading to cleavage of the inhibitor as a substrate producing free E and Is, a reactive site-cleaved inhibitor. EI* formation for serpin-proteinase reactions have been proposed to involve a structural rearrangement within the inhibitor induced by binding to the enzyme (13, 33). If, for a fraction of its encounters with the inhibitor, a proteinase is capable of hydrolyzing the P1-P1’ bond before the conformational change is complete, the SI for the reaction will be greater than 1(33). The second scheme (Scheme 2) is based on the studies of Declerck et al. (34) which suggest that a single serpin may have interconvertible inhibitor (Ia) and substrate (Ib)conformations. The value SI would then depend on the relative rates of chymase interaction with each conformer and the position of the conformer equilibrium. E
+I
k3
kl
E.1- EI* k-1
lkz I’+E
SCHEME1
value was about 10-fold lower than rACT-L358W, which had an SI close to 1, and 4-fold lower than rACT-L358M, which had an SI of 2. The order of k’/[I] values for three P1 variants was Met > Leu > Phe (total range was 2-fold). This order was opposite that observed for chymotrypsin (total range was 5-10-fold) with the same variants (10, 11). The variation of rACT inhibitor properties as afunction of P1 substitution may be rationalized by either of two schemes.
E
+ Ia e E.1 + El’ SCHEME 2
The order of SI values (Phe > Leu > Met > Trp) as a function of P1 substitution qualitatively paralleled the substrate preference of chymase (Phe > Leu > Met W) toward
-
Reaction of Human Chymase with Variant Antichymotrypsins peptide-pNA substrates as measured by $.t or %at/Km values (Table 111).This correlationis consistent with the notion that the substratepathway in eitherScheme 1or Scheme 2 is more sensitive to P1 substitution than is the inhibitor pathwayand suggests a parallelism for the effects of P1 substitution on SI values and on the rate determining step(s) for peptide-pNA hydrolysis. Similar agreement, however, was not observed for chymase catalysis of PI-substituted angiotensin I analogs where hatand kat/Kmvalues were reported to vary in the order of Phe Trp > Leu (27). The difference observed for the two simple sets of substrates might reflect differences in the natureof the rate-determining stepsfor chymase catalyzed hydrolysis of each substrate type (35). Partitioning occurs as two first-order reactions in Scheme 1and astwo second-order reactions in Scheme 2. AS a result, the naive expectation is that in Scheme 1, SI-1 values (rate of the substrate pathway reaction relative to the rate of the inhibitor pathway)may parallel kcatvalues, whereas in Scheme 2 they may parallel kat/Kmvalues. Quantitatively, relative SI1 values (Phe:Leu:Met:Trp) for P1 variants agree better with katthan k&Km values of peptide-pNA substrates; however, the results do not permit a clear choice between Schemes 1 and 2. The result of P1 substitutions on k'/[I] values showed that these values decreased as SI values for corresponding variants increased (Table I). Although this result for P1 variants is a possible consequence of the branch points in Scheme 1 (the partitioning of E . I by reaction steps with rate constants k2 and k3) and Scheme 2 (1a:Ib equilibrium), the low k'/[I] and low SI values obtained for the rACT-P3P3' variant demonstrate that these two parameters do not necessarily vary inversely. rACT-P3P3' is a more extensively substituted variant compared with P1 variants. The greater degree of change to thereactive loop of this rACT is likely an factor influencing its behavior. The order of k'/[I] values for inhibition of chymase by Met, Leu, and Phe variants was opposite that observed for inhibition of chymotrypsin (Phe > Leu > Met) by the same variants (10, l l ) , despite bothproteases having the same general substrate preferences (3, 36). In contrast to chymase, the SI value for the interaction of chymotrypsin with each of these variants is close to 1 (10, 11), suggesting that suppression of the substrate pathway may account for the observed difference in the order of inhibition rate constants. In summary, the high SI observed for the interaction chymase with ACT and its wide variation in mutants allow for the analysis of serpin-proteinase interactionsin a manner not available for other target proteinases. Our results, however, do not provide definitive evidence permitting a clear choice between the branched orconformer model to explain SI values greater than 1. Nevertheless these studies demonstrate the importance of including a rapid partitioning scheme in the general equation for serpin-proteinase interactions and the need for further defining the mechanism producing high SI values.
-
Acknowledgments-We
are thankful to thePathology Department
23633
at the University of Pennsylvania and to the National Disease Research Interchange for collection of human skin used in the purification of human chymase. Weare grateful to Nora Zuiio, Pirjo Tuominen, and Michael Lanuti for assistance in purification of inhibitors and chymase. We thank Xuzhuo Liu for construction of several variant inhibitors, and Quihui Zhong for making the rACTL358W variant. Special thanks to Michael Plotnick and to Darrell R. McCaslin (Department of Chemistry a t Rutgers University) for many helpful discussions. We also thank William Witmer, the Department of Dermatology at the University of Pennsylvania, for assistance in preparing illustrations. Protein sequencing was performed at theWistar Institute Protein Microchemistry Core Facility, Philadelphia PA. REFERENCES 1. Schechter, N. M.,Choi, J. K., Slavin, D. A., Deresienski, D. T., Sayama, S., Dong, G., Lavker, R. M., Proud, D., and Lazarus, G. S. (1986) J. Immunol. 137,962-970 2. Sayama, S., Iozzo, R. V., Lazarus, G. S., and Schechter, N. M. (1987) J. Biol. Chem. 262,6808-6815 3. Powers, J. C., Tanaka, T., Harper, J. W., Minematsu, Y., Barker, L., Lincoln, D., Crumley, K. V., Fraki, J. E., Schechter, N. M., L a m s , G. L., Nakajima, K., Nakashino, K., Neurath, H., and Woodbury, R. G. (1985) Biochemistry 24,2048-2058 4. Schechter, N. M., Sprows, J. L., Schoenberger, 0. L., Lazarus, G. L., Cooperman, B. S., and Rubin, H. (1989) J. Biol. Chem. 264, 2130821315 5. Travis, J., and Salvesen, G . S. (1983) Annu. Reo. Biochem. 6 2 , 655-709 6. Carrell, R. W., and Boswell, D. R. (1986) Res. Mongr. Cell Tissue Physiol. 12,403-418 7. Beatty, K., Bieth, J., and Travis, J. (1980) J. Biol. Chem. 266,3931-3934 8. Schechter, I., and Berger, A. C. (1967) Biochem.Bwphys. Res. Cornmun. 2 7 , 157-162 9. Cooperman, B. S., Stavridi, E., Nickbarg, E., Rescorla, E., Schechter, N. M., and Rubin, H. (1993) J. Bwl. Chem. 268,23616-23625 10. Rubin, H., Wan , Z , Nickbarg, E. B., Mclarney, S., Naidoo, N.! Schoenberger, 0. L., fohnson, J. L., and Cooperman, B. S. (1990) J. Bwl. Chem. 265,1199-1207 11. Kil atrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z.-m., Clifford, T. F., Janach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146,2388-2393 12. Schultz, A. J. Baumann, U., Knof S., Jaeger, E., Huber, R., and Laurell, C. B. (1991j Eur. J. Biochem. 194,51-56 13. Skriver, K., Wikoff, W. R., Pataton, P. A., Tausk, F., Schapira, M., Kaplan, A. P., and Bock, S. C. (1991) J. Bwl. Chem. 266,9216-9221 14. Bock, S. C. (1991) Protein Eng. 4.221-221 15. Stein, P. E., Andrew, G. W. L. Finch, J. T., Turnell W. G., McLaughlin, P. J., and Carrell, R.W. (1960) Nature 347,99-162 16. Schechter, N. M., Slavin, D., Fetter, R. D., Lazarus, G. L., and Fraki, J. A. (1988) Arch. Biochem. Bwphys. 262,232-244 17. Schechter, N. M., Iranl, A.-M. A,, Sprows J. L., Abernethy, J., Wintroub, B., and Schwartz, L. B. (1990) J. Immuhl. 1 4 6 , 2652-2661 18. Schechter, N. M.(1990) Mongr. Allergy 2 7 , 114-131 19. Schonbaum, G. R., Zerner, B., and Bender, M. L. (1961) J. Biochem. 2 3 6 , 2930-2935 20. Chase, T., Jrand Shaw, E. (1970) Methohods Enzymol. 1 9 , 20-27 21. Petersen, L. C., and Clemmensen, I. (1981) Biochem. J. 1 9 9 , 121-127 22. SDeicher. D. W. (1989) in Techniolles m Protetn Chemrstn, (Hueli. T. E.. . ~ ~ . ~ ed) pp: 24-35, Academic Press, Diego 23. Morii, M.,Odano, S., and Ikenaka, T. (1979) J . Biochem. 86,915-921 24. Boswell. D. R.. JeDDSSOn. J.-0.. Brennan. S. 0.. and Carrell. R. W. (1983) Bioc6im. Bwphji. Acta 744,'212-218 25. Baumann, U., Huber, R., Bode, W., Grosse, D., Lesjak, M., and Laurell, C. B. (1991) J. Mol. Biol. 218,595-606 26. Patston, P. A,, Gettins, P.,Beechem, J., and Schapira, M. (1991) Biochemistry 30,8876-8882 27. Kinoshita, A., Urata, H., Bumpus, F. M., and Husain, A. (1991) J. Biol. Chem. 2 6 6 , 19192-19197 28. Reilly C. F., Tewksbury, D. A,, Schechter, N. M., and Travis, J. (1982) J. Bioi. Chem. 267,8619-8622 , S., Kaempfer, C. E., and 29. Wintroub, B. U., Schechter, N. M., L ~ Z ~ N SG. Schwartz, L. B. (1984) J. Inuest. Dermutol. 83,336-339 30. Jenne. D. E.. and TschoDo. d . (1991) Aiochem. J. 276.567-568 .. 31. Bjork,'I, and Fish, W. W. (1982) J.'Biol. Chem. 26719487-9493 32. Olson, S. T. (1985) J. Biol. Chem. 260,10153-10160 33. Biork. I.. Nordling. K.. Larsson. I.. and Olson. S. T. (1992) , , J. Biol. Chem. - 2 6 7 , 19047-19650 ' 34. Declerck, P. J., De Mol, M., Vaughan, D. E., and Collen, D. (1992) J. Biol. Chem. 2 6 7 , 11693-11696 35. Stein, R. L., and Strimpler, A. M . (1987) Biochemistry 2 6 , 1301-1305 36. Schellenberger, V., Braune, K., Hopmann, H.-J., and Jakuhke,H.-D. (1991) Eur. J.Biochem. 199,623-636 ~
~.
&an ~~~
~~~~
~
~
~
~~~~
~~~~
_.~..~
~
~"~~ ~~
,
I
I
.
I
I
