active site mapping of human and rat urinary kallikreins by peptidyl chloromethyl ketones

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 202, No. 2, July, pp. 420-430, 1980

Active Site Mapping of Human and Rat Urinary Kallikreins by Peptidyl Chloromethyl Ketones’

CHARLES KETTNER,* CHRISTOPHER MIRABELLI,* JACK V. PIERCE,t AND ELLIOTT SHAW*

*Biology Department, Brookhaven National Laboratory, Upton, New York 11973 and THeart, Lung, and Blood Institute, National Institutes of Health,

Bethesda, Maryland 20014

Received December 21. 1979

The reactivity of human and rat urinary kallikrein has been determined with peptides of arginine and lysine chloromethyl ketone. Pro-Phe-ArgCH,Cl, the reagent corresponding to the sequence of kininogen hydrolyzed by kallikrein, was considerably more effective than reagents containing other substituents in the P, and PZ positions (the Arg and Phe binding sites, respectively). Pro-Phe-ArgCH,Cl inactivates the human enzyme at the 10m5 M level (Ki 45 pM, kl 0.36 min-‘) and the rat enzyme at the 10-&M level (K, 4.8 pM, k2 0.26 min-I). More effective reagents were obtained by substitution of D-Phe for the P3 Pro and addition of a dansyl residue in the P, position, yielding reagents effective at the lo-‘M level for both kallikreins. Expansion of the sequence of kininogen to accomodate the P, and P, binding sites of kallikrein resulted in a reagent, Phe-Ser-Pro-Phe-ArgCH&l, which is approximately B-fold more reactive than the corresponding tripeptide analog for human kallikrein, while for the rat kallikrein, the tri- and pentapeptide analogs are comparable in reactivity. The importance of Arg in the P, position and Phe in the P2 positions in the sequence of kallikrein’s physiological substrate in determining specificity was shown by comparison of the reactivities of the proteases with Ala-Phe-ArgCH,Cl and Ala-Phe-LysCH,Cl and with Pro-Phe- ArgCH$l and Pro-Gly-ArgCH,Cl. Substitution of Lys for the P, Arg and substitution of Gly for the P, Phe decreased the reactivity of the reagent lo- and 150-fold, respectively, for the human kallikrein and 200- and 250-fold, respectively, for the rat kallikrein. Sub- stitution of L-amino acid residues for the P, Pro had little effect on the reactivity of human kallikrein with the affinity labels and decreased the reactivity of the rat enzyme with the affinity labels from 3- to &fold.

This communication describes the results in the liberation of the C-terminal of of an extension of our studies on affinity bradykinin for the former (4) and Lys- labeling of trypsin-like proteases to gland- bradykinin for the latter (5). ular kallikreins. Kallikreins from the kidney, Selective affinity labels for glandular pancreas, and submaxillary gland are kallikreins will be useful in either deducing similar in their physical properties and in or confirming their physiological roles. their reactivities with substrates and in- Protein substrates for glandular kallikreins hibitors (1, 2>, but differ markedly from other than kininogen only recently have plasma kallikrein (3). Both plasma kallikrein been identified. Ole-Moi et al. (6, ‘7) have and glandular kallikreins, however, share a shown that their purified pancreatic kalli- common specificity for the same bond of krein in concert with carboxypeptidase kininogen substrates which is hydrolyzed converts proinsulin to a product with the

same electrophoretic mobility as insulin L This work was supported by NIH Contract and have suggested that kallikrein func-

l-YOl-HV-‘70021-00 and by the U. S. Department of tions in the in vitro conversion of proinsulin Energy. to insulin. The mouse submaxillary gland

0003~9861/80/080420-11$02.00/O 420 Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

AFFINITY LABELING OF URINARY KALLIKREINS 421

contains three proteases, p-nerve growth factor-endopeptidase, nerve growth factor y-subunit, and epidermal growth factor- binding protein, which exhibit significant levels of kininogenase activity. The former, which removes the N-terminal octapeptide from the p-nerve growth factor (9), appears to be identical to submaxillary kallikrein whereas the latter proteases, whose apparent function is the activation of the growth factors through limited proteolysis, are similar to submaxillary gland kallikrein in physical and immunological properties. Of particular interest is the correlation between low levels of urinary kallikrein and essential hypertension (11). Evidence has been presented that purified human urinary kallikrein acts to convert prorenin to renin (12, 13), but this reaction has not been demonstrated in vivo and the role of renal kallikrein in renal function remains unclear.

Recently, we prepared an affinity label corresponding to the amino acid sequence of kininogen hydrolyzed by kallikrein in the liberation of the C terminal of bradykinin (14). The reactivity of this reagent, Pro-Phe-ArgCH,Cl, in the inactivation of plasma kallikrein demonstrated that a selective affinity label could be obtained for an individual trypsin-like protease by the synthesis of a chloromethyl ketone corresponding to the amino acid sequence of its physiological substrate. Pro-Phe- ArgCH,Cl readily inactivates plasma kallikrein at the lo-* M level, distinguishing it from other trypsin-like proteases, plasmin, thrombin, urokinase, and factor Xa. Sim- ilarily, this approach has been success- fully applied in the preparation of selective affinity labels for thrombin (15, 16) and factor Xa (unpublished observation). These results have indicated that binding in the subsites of plasma trypsin-like proteases which function in regulatory roles through limited proteolysis is important in determining the selectivity of these enzymes for their physiological substrates.

This study evaluates the reactivity of Pro-Phe-ArgCH,Cl with glandular kallikrein from two species, human and rat. The reactivity of the tripeptide analog is compared with di-, tetra-, and pentapeptide

analogs corresponding to the sequence of kininogen to determine the influence of binding in the second through fifth subsites. The reactivities of the urinary kallikreins were also determined with affinity labels corresponding to the amino acid sequence of physiological substrates for other proteases to obtain additional information on the specificity of these proteases. Further- more, progress in the design of more effective affinity labels for the glandular kallikreins is described.

MATERIALS AND METHODS

Affinity labels. Peptides of arginine, homoarginine (Har),* and lysine chloromethyl ketone were prepared by coupling N-blocked peptides to either H-Arg(NO,)CH,Cl (14), H-Har(NO,)CH,Cl (17), or H-Lys(Z)CH,Cl (14) using the mixed anhydride procedure we have described previously (14). Final products were obtained by removing protecting groups with anhydrous HF. The syntheses of reagents specifically prepared as affinity labels for the kallikreins and their characterization are described in the supplementary material. The syntheses of Ala-Phe- ArgCH,Cl, Ala-Phe-LysCH,Cl, and Pro-Phe- ArgCH,Cl, were described in Ref. (14), Phe-Ala- LysCH,CI and Ile-Leu-ArgCH,Cl in Ref. (17), Glu-Gly-ArgCH,Cl in Ref. (18).

Kinetic studies. HUK (1’7 Tos-Arg-OMe unit& mg) was prepared by a modification of the immuno- precipitation procedure of Pierce (19) using a mono- specific antibody to HUK and RUK (50 Tos-Arg-OMe units/mg) was prepared by the procedure of Chao et al. (20). Inactivation reactions of HUK and RUK were conducted at 25°C in 5.00 ml of 50 mM Pipes buffer, pH 7.0, which was 0.20 M in NaCl. Inactivation reactions were initiated by adding either 0.0125 Tos-Arg-OMe unit of HUK or 0.016 Tos-Arg- OMe unit of RUK to solutions of the chloromethyl ketones. From six to eight 0.50-ml aliquots were subsequently removed at 3-min intervals and were added to 1.50 ml of 0.20 M Tris buffer, pH 8.0, containing 0.20 M NaCl, Z-Lys-SBzl as a substrate (21) and 5,5’-dithiobis(2-nitrobenzoic acid) as a chromogen. The final concentrations of substrate

* Abbreviations used: HUK, human urinary kallikrein; RUK, rat urinary kallikrein; Dns, N,N- dimethylamino-naphthalene-5-sulfonyl; Har, homoarginine; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); Z-Lys-SBzl, thiobenzyl carbobenzoxylysinate; Tos-Arg-OMe, tosylarginine methyl ester; AC, acetyl.

3 Tos-Arg-OMe unit, the quantity of protease which will hydrolyze 1.0 pmol of substrate at pH 8.0 and 30°C in 1 min.

422 KETTNER ET AL.

TABLE I

KINETIC CONSTANTS FOR THE INACTIVATION OF HUMAN URINARY KALLIKREIN BY SUBSTRATE-DERIVED CHLOROMETHYT, KETONEB

Affinity labels W4.k,lK, Ki (M-l min-I) (PM)

k, (min-‘)

I Ac-Phe-ArgCH,Cl 0.81 56.0 0.46 II Pro-Phe-ArgCH,Cl 0.79 45.0 0.36 III Ac-Pro-Phe-ArgCH,Cl 0.34 120 0.41 IV Ser-Pro-Phe-ArgCH,Cl 0.65 180 1.20 V Phe-Ser-Pro-Phe-ArgCH,CI 4.20 21.2 0.89

n Kinetic constants were measured at 25°C in 50 mM Pipes buffer, pH 7.0, which was 0.20 M in NaCl. The bimolecular constant, k,lK,, was determined by the method Kitz and Wilson (22) and values of Ki, the reversible disassociation constant, were determined by the method of Lineweaver and Burk (23). The tirst order rate constant for the inactivation reaction, kl, was calculated from Ki and k,lKi. For specific details see -, Materials and Methods.

and chromogen were 0.30 and 0.33 mM, respectively. Thioesterase activity was determined in a continuous assay for 2 to 3 min on a Beckman 5230 spectrometer using a recorder scale of 0.05 absorbance unit and a chart speed of 1 in./min. Under these conditions 0.0012 and 0.0016 Tos-Arg- OMe unit of HUK and RUK, respectively, yielded a linear change in absorbance of 0.035 to 0.040 absorbance unit during a 3-min assay. In the evaluation of the reactivity of the proteases with the affinity labels, no inactivation was observed during the thioesterase assay.

The concentration dependence of the inactivation reactions were determined with at least five concentrations of affinity labels such that the half-times for inactivation were in the range of 6 to 18 min. The concentration ranges of affinity labels used in determining values of kJKi reported in Table I were: for compound I, 5.5-12.5 PM; II, 9.5-18.2 PM; III, 8.2-21.0 pM; IV, 6.9-14.3 PM; and V, 0.80-2.0 PM. Kinetic constants, Ki, k,, and kJKi, were determined from double reciprocal plots of k,,, vs concentration of affinity label according to the procedure described by Kitz and Wilson (22).

Values of K, for HUK were determined by the procedure of Lineweaver and Burk (23) since in several eases double reciprocal plots of k.,, vs in- hibitor concentration yielded intercepts too close to the origin to be considered a reliable indication of saturation kinetics. At least seven rates for the initial hydrolysis of Z-Lys-SBzl over a concentration range from 0.10 to 0.50 mM were measured at 25°C in 2.00 ml of 50 mM Pipes buffer, pH 7.0, containing 9.20 M NaCl, 0.33 mM 5,5’-dithiobis(2-nitrobenzoic acid), and 5.05 x 10m3 Tos-Arg-OMe unit of HUK both in the presence and absence of affmity label. The following concentrations of affinity labels were used in determining the values of Ki for HUK re-

ported in Table I: I, 0.136 mM; II, 0.0178 mM; III, 0.210 mM; IV, 0.103 mM; andV, 0.0176 mM. Values of k, were calculated from K( and from kJKi.

Bioassays. Bioassays of RUK were conducted essentially by the procedure described by Ohanian et al. (24). In a typical assay, a 400-~1 sample of RUK in phosphatebuffered saline, pH 7.4, was injected in the right carotid artery of Dahl hypertension-resistant rats anesthetized with urethane (1.6 g/kg). Blood pressure was monitored by a Grass polygraph and a Statham pressure transducer connected to a cannula in the left femoral artery. Samples of RUK (2 wg) were treated with either D- Phe-Phe-ArgCH,Cl, Pro-Phe-ArgCH,Cl, or Phe- Ser-Pro-Phe-ArgCH,Cl at 25°C and pH 7.4 and after 30 min, the hypotensive response to the samples were determined. The hypotensive response to RUK was compared with the response to RUK inactivated with the chloromethyl ketones and with the response to the chloromethyl ketones, treated in a manner analogous to the samples of inactivated RUK.

In vitro kininogenase activity was determined in Dr. H. S. Margolius’ laboratory, Department of Pharmacology, Medical University of South Carolina, for both RUK and HUK by the procedure of Shimamoto et al. (25, 26) in which liberated kinin was measured by radioimmunoassay following incubation with purified bovine low molecular weight kininogen. The affinity labels were incubated with RUK (0.01 Tos-Arg-OMe unit) or with HUK (0.05 Tos-Arg-OMe unit) for 30 min at 25°C in 0.50 ml of phosphate-buffered saline, pH 7.4. An aliquot (0.050 ml) was removed and diluted to 0.50 ml with 0.10 M

phosphate buffer, pH 8.5, containing 1.5 pg of kininogen, 3.6 mM phenanthroline, and 30 mM EDTA. After incubating 20 min at 37”C, the reaction was stopped by boiling 10 min and liberated kinin was determined by radioimmunoassay. In the


absence of affinity label, RUK and HUK exhibited kininogenase activities of 5.0 x lo5 and 1.0 x 10” pg bradykinin/20 min/Tos-Arg-OMe unit, respectively, under the conditions of these assays.

RESULTS

Reactivity of Human Urinary Kallikrein with Reagents Corresponding to its Physiological Substrate

Initially in the study of human urinary kallikrein, peptidyl arginine chloromethyl ketones of varying length were synthesized which correspond to the amino acid sequence of kininogen hydrolyzed by kallikrein in the liberation of the C terminus of bradykinin. The reactivity of these reagents with HUK were measured to eval- uate the influence of binding in the P2-P, positions on reagent effectiveness. The reversible binding constant (K,), the first- order rate constant for the irreversible step of the reaction (ZcJ, and the bimolecular constant (k,/KJ for the affinity labeling mechanism, Eq. [l],

k2 E + I * EI + alkylated protease, ]ll

are shown in Table I for the substrate analogs. Bimolecular constants, kz/Ki, were determined from the concentration dependence of the inactivation reaction according to Eq. [2] where k,,, is the apparent, first-order rate constant for inactivation and Z is the concentration of affinity label (22):

1 -= 5l+’ k k, I k, *

[21 BPP

In several eases, the intercept of double reciprocal plots were too close to the origin for accurate evaluation of k,. Therefore, values of Ki were determined from initial velocity studies in which the hydrolysis of substrate was measured in the presence of affinity label and data were analyzed by the method of Linweaver and Burk (23). Values of k, were then calculated from k,lKi and Ki. Hydrolysis of substrate in the presence of affinity label was linear

over a sufficient time interval to allow the determination of initial velocity prior to inactivation in the assay.

As shown by the values of kslKi in Table I for the inactivation of HUK, Ac- Phe-ArgCH,Cl, Pro-Phe-ArgCH,Cl, and Ser-Pro-Phe-ArgCH,Cl are almost identical in their reactivity, while diminished reactivity was observed for Ac-Pro-Phe- ArgCH,Cl. Examination of the contribu- tion of the two steps in the affinity labeling mechanism revealed that the comparable reactivity of Ac-Phe-ArgCH,Cl and Pro-Phe-ArgCH,Cl was due to almost identical binding constants and the lower reactivity of Ac-Pro-Phe- ArgCH,Cl is due to a twofold increase in Kj. On the other hand, the affinities of HUK for Ser- Pro-Phe-ArgCH,Cl and Ac-Pro-Phe- ArgCH,Cl are almost identical and the greater reactivity of the tetrapeptide analog is due to a larger rate constant for the alkylation reaction. Phe-Ser-Pro- Phe-ArgCH,Cl is fivefold more reactive than the corresponding di- and tripeptide analogs. This difference is due to a larger value of k, and a lower value of Ki.

The Reactivity of HUK with Tripeptide Analogs

The reactivity of HUK with the arginine chloromethyl ketone corresponding to the sequence of its physiological substrate, Pro- Phe-ArgCH,Cl (II), was compared with its reactivity with tripeptide analogs whose sequences are not related to its physiological substrate to determine the influence of amino acid substitution on reagent effectiveness. Reagents reported in Table I are also shown in Table II alone with a series of additional tripeptide analogs ar- ranged in decending order of their effectiveness for the inactivation of HUK with the concentrations of reagents and half-times (t,,,) for pseudo-first-order inactivation. Values of k,,JZ were calculated for comparison of the reactivity of the reagents using the relationship in Eq. ]31 (2%

k k mp = __1 Z Ki ’

424 KETTNER ET AL.

TABLE II

THE SUSCEPTIBILITY OF URINARY KALLIKREINS TO INACTIVATION BY PEPTIDES OF ARGININE CHLOROMETHYL KETONES

Affinity label

p, p4 p3 p, p,

Human urinary kallikrein Rat urinary kallikrein

Concen- Concen- tration t,,2 10-4~k.,,iIc tration t,,, lo-‘.k,,,,lI

(PM) (min) (M-l min-‘) (Pm (min) (M-l min-‘)

s Inactivations were conducted at 25°C in 50 mM Pipes buffer, pH 7.0, which was 0.20 M in NaCl. b t112 is the half-time for pseudo-first-order inactivation at the indicated concentration of affinity label. c k,,,/I, the ratio of the apparent, pseudo-first-order rate constant for inactivation (k,,) to the concentration of affinity

label (I), is an estimate of the bimolecular constant, k,lKi. These values were calculated from the concentrations of affinity labels and tl,* (k,,, = In 2/t,,,) from the data shown except for reagents I-V in which actual values of ktlKi are reported.

I Ac-Phe-ArgCH,Cl 5.0 17.6 0.81 II Pro-Phe-ArgCH,Cl 5.0 23.8 0.79 III Ac-Pro-Phe-ArgCH,Cl 10.0 19.2 0.34 IV Ser-Pro-Phe-ArgCH,Cl 5.0 28.3 0.65 V I ‘he-Ser-Pro-Phe-ArgCH$l 0.75 22.5 4.2 VI u-Phe-Phe-ArgCH,Cl 0.10 37.4 18 VII Dns-Pro-Phe-ArgCH,Cl 0.63 15.5 7.1 VIII Dns-Glu-Phe-ArgCH,Cl 2.0 26.6 1.3 IX Phe-Phe-ArgCH,Cl 4.0 27.5 0.63 X Ala-Phe-ArgCH,Cl 10 20.2 0.34 XI Glu-Phe-ArgCH,Cl 10 21.9 0.32 XII D-Phe-Pro-ArgCH,Cl 30 8.6 0.27 XIII Dns-Glu-Gly-ArgCH,Cl 20 19.7 0.18 XIV Ile-Leu-At&H&l 50 18.0 0.077 xv Pro-Phe-HarCH,Cl 200 11.1 0.031 XVI Ala-Phe-LysCH,Cl 250 13.8 0.020 XVII Val-Pro-ArgCH,Cl 500 35.5 0.0039 XVIII Gly-Val-ArgCH,CI 1000 22.6 0.0031 XIX Pro-Gly-ArgCH,Cl 1000 24.2 0.0029 xx Ile-Pro-ArgCH,Cl 500 49.2 0.0028 XXI Phe-Ala-ArgCH,Cl 1000 33.8 0.0021 XXII Val-Val-ArgCH,Cl 4000 10.6 0.0016 XXIII Ala-Lys-ArgCH,Cl 1000 44.6 0.0016 XXIV Glu-Gly-ArgCH,Cl 5000 15.2 0.00091 xxv Ae-Gly-Gly-ArgCH,Cl 6700 18.8 0.00055 XXVI Ile-Glu-Gly-ArgCH,Cl 5000 30.7 0.00045

I 1.0 21.3 3.9 II 1.0 14.3 5.5 III 2.0 23.3 1.4 IV 1.0 21.4 3.4 V 0.50 17.8 6.9 VI 0.10 25.8 27.0 VII 0.10 25.4 27.0 VIII 2.0 22.2 1.6 IX 2.5 22.5 1.2 X 2.0 22.8 1.5 XI 1.5 26.5 1.7 XII 5.0 20.8 0.67 XIII 16 31.7 0.14 XIV 5.0 20.6 0.67 xv 1000 22.2 0.0031 XVI 500 17.9 0.0077 XVII 50 14.6 0.095 XVIII 100 17.8 0.039 XIX 200 19.4 0.018 xx 100 17.5 0.040 XXI 100 14.6 0.048 XXII 100 24.0 0.029 XXIII 406 19.2 0.0090 XXIV 100 23.9 0.029 XXV 300 20.0 0.012 XXVI 300 21.4 0.011

if I << Kim [31

The effect of substitution of Lys and homoarginine for the P, Arg is shown by comparison of the reactivity of Ala-Phe- ArgCH,CI (X) with Ala-Phe-LysCH,Cl- (XVI) and by comparison of the reactivity of Pro-Phe-ArgCH,Cl(II) with Pro-Phe- HarCH,Cl (XV). In each case, the arginine chloromethyl ketone is lo-fold more reactive. A considerably larger. difference in reactivity was observed when Gly was substituted for Phe in the P, position. This is shown by the EO-fold larger value of k,,JZ for Pro-Phe-ArgCH,Cl(II), than for Pro-

Gly-ArgCH&l (XIX). No significant difference was observed in the reactivity of HUK with reagents containing Pro, Ala, Glu, or Phe in the P3 position shown by comparison of values of k,,JZ for Pro- Phe-ArgCH,Cl (II), Ala-Phe-ArgCH,Cl- (X), Glu-Phe-ArgCH,Cl(XI), and Phe- Phe-ArgCH,Cl (IX).

A survey of reagents VI-XXVI further illustrates the importance of binding in secondary sites on the reactivity of HUK. This is shown by a range in reactivity of three orders of magnitude from the more effective reagents to the least reactive ones.

. n--\r-mTr I . v.Y.7 TllT,-. rrn Al”! INl’I’Y LAJSl!,LINb UP URINARY KALLIKREINS 425

A notably enhanced reactivity was observed for reagents with D-Phe in the P3 position and Dns in the P, position. HUK is 70- to go-fold more reactive with n-Phe- Pro-ArgCH,Cl (XII) than analogs containing Val and Ile in the P, position (reagents XVII and XX). The preference of HUK for Dns in the P, position is shown by the 180- fold greater reactivity of Dns-Glu-Gly- ArgCH,Cl (XIII) than Glu-Gly-ArgCH,Cl (XXIV). Subsequently, D-Phe-Phe-Arg- CH,Cl(VI) and Dns-Pro-Phe-ArgCH,Cl (VII) were synthesized in the expectation of obtaining improved reagents. D-Phe- Phe-ArgCH,Cl (VI) proved to be the most effective affinity label for HUK inactivating it at the lo-'M level. Addition of Dns to Pro-Phe-ArgCH,Cl increased its reactivity by greater than lo-fold, but this increase was smaller than expected. DNS- Glu-Phe-ArgCH,Cl(VIII) was then prepared in the hope of obtaining a reagent of enhanced reactivity, as resulted on the addition of Dns to Glu-Gly-ArgCH,Cl, but the addition of Dns- to Glu-Phe- ArgCH,Cl only increased reactivity 3-fold.

k&, indicates that less than a twofold difference exists in the reactivity of di-, tri-, tetra-, and pentapeptide analogs with RUK except for Ac-Pro-Phe-ArgCH,Cl (III) which is two- to fivefold less reactive than the other reagents. Values of k2 measured for the inactivation of reagents I-V were 0.39, 0.26, 0.32, 0.24, and 0.25 min-’ , respectively, showing that values of kz/Ki in Table II are indicative of the affinity of RUK for the arginine chloromethyl ketones.

The large differences in the reactivity of HUK with these reagents most probably reflect differences in the affinity of the protease for the affinity label. This has been the case for a number of proteases examined with tripeptide chloromethyl ketones (14, 15, 18) and is true for three of the five inhibitors shown in Table I. The larger values of Ic, observed for the tetra- and pentapeptide analogs are exceptional; however, these differences are relatively small.

As described for HUK, the reactivity of RUK toward tripeptide analogs in which single amino acid residues are varied in the P1, Pz, and P, binding sites of Pro-Phe-ArgCH,Cl (II) was determined. The greater reactivity of RUK with reagents containing Arg in the P, position is shown by the 200-fold greater value of k&Z for Ala-Phe-ArgCH,Cl (X) than for Ala-Phe-LysCH,Cl (XVI) and the lOO- fold greater value for Pro-Phe-ArgCH,Cl (II) than for Pro-Phe-HarCH,Cl (XV). The effect of Phe in the P, position is shown by the 300-fold greater reactivity of Pro-Phe-ArgCH,Cl (II) with RUK than Pro-Gly-ArgCH,Cl (XIX). In contrast to the much greater reactivity of RUK with reagents containing Arg in the P, position and Phe in the P, position, considerably smaller differences were obtained when Ala, Glu, and Phe were substituted for Pro in the P, position. This is shown by the three- to fivefold greater reactivity of RUK with Pro-Phe-ArgCH,Cl (II) than with Ala-Phe-ArgCH,Cl (X), Glu-Phe- ArgCH&l (XI), or Phe-Phe-ArgCH,Cl (IX).

Reactivity of Rat Urinary Kallikrein with Peptidyl Chloromethyl Ketones

D-Phe-Phe-ArgCH,Cl (VI) and Dns- Pro-Phe-ArgCH,Cl (VII) were the most effective affinity labels for RUK, both inactivating the protease at 1.0 x lo-’ M.

The reactivity of arginine chloromethyl ketones was also determined with rat urinary kallikrein (RUK). Kinetic constants, Ki and k,, were determined for those reagents corresponding to the sequence of kininogen (reagents I-V) and values of k,l Ki are shown in Table II with typical concentrations of reagents and half-times for inactivation. Comparison of the bimolecular constants for inactivation of RUK,

Biological Assay of Urinary Kallikrein

The foregoing results demonstrated that the arginine chloromethyl ketones are acting in an irreversible manner in inactivating both RUK and HUK, however, these results are based on the esterase activities of the proteases. In further characterizing the reactivity of arginine

426 KETTNER ET AL.

chloromethyl ketones with the urinary kallikreins, we have shown that the reagents inactivate the biological activity of RUK by demonstrating that they block the ability of RUK to liberate Lys-bradykinin from rat kininogen in viva with a resultant lowering of arterial blood pressure.

Samples of RUK were incubated for 30 min with D-Phe-Phe-ArgCH,Cl, Pro- Phe-ArgCH,Cl, and Phe-Ser-Pro-Phe- ArgCH,Cl at levels of reagent sufficiently high to completely inactivate the protease’s esterase activity. Concentrations used are fivefold the level shown in Table II. The hypotensive response of rat arterial blood pressure to RUK after reacting with the arginine chloromethyl ketones is compared with that of the untreated protease in Fig. 1. After incubation with the affinity labels, the biological activity of RUK was lost since the response obtained from an incubation mixture consisting of RUK treated with the chloromethyl ketone was no greater than that of the chloromethyl ketone alone.

The time dependence of the inactivation reaction of RUK has been determined for one reagent. D-Phe-Phe-ArgCH,Cl at 2.5 x 10e7 M was incubated with RUK and portions consisting of 2 rug of RUK were removed at timed intervals and assayed. After 10 min, the hypotensive response was reduced to that produced by 1 pg of RUK indicating approximately 50% inactivation. This rate of inactivation cor- responds to the rate observed with the esterase assay (Table II).

Similar in viva experiments were not possible for HUK, probably because rat kininogen is a poor substrate for it. How- ever, the kininogenase activities of both proteases were inactivated as demonstrated by radioimmune assay for kinin following their action on purified bovine, low molecular weight kininogen. In these experiments, HUK was incubated for 30 min at 25°C with 10 PM Pro-Phe-ArgCH,Cl, 5 PM Phe-Ser-Pro-Phe-ArgCH&l, and 0.5 PM D-Phe-Phe-ArgCH,Cl reducing kininogenase activity to 1, 11, and 3%, respectively, of the control. Similarily, when RUK was allowed to react with the

FIG. 1. The response of rat arterial blood pressure to injections of RUK following treatment with affinity labels. RUK (2 Fg) was allowed to react with either 0.50 pM D-Phe-Phe-ArgCH,Cl (VI), 10 pM Pro- Phe-ArgCH,Cl (II), or 2.5 pM Phe-Ser-Pro- Phe-ArgCH,Cl (V) for 30 min at 25°C and pH 7.4; the response of blood pressure to the entire sample (400 ~1) was determined. Arrows indicate the time in which samples were injected and numbers indicate the composition of the sample as follows; (1) RUK, (2) RUK + VI, (3) RUK, (4) RUK + II, (5) RUK, (6) RUK + V, (7) RUK, (8) VI. Sampies of II and V alone gave responses almost identical to VI.

chloromethyl ketones at the levels described in Fig. 1, no kininogenase activity could be detected.

DISCUSSION

We have expanded our studies of the affinity labeling of trypsin-like proteases to glandular kallikreins in order to obtain inhibitors of this group of proteases which may be useful in clarifying their physiological functions. These studies have yielded a number of reagents which are highly effective in the inactivation of both HUK and RUK. For example, D-Phe-Phe- ArgCH$l and Dns-Pro-Phe-ArgCH,Cl readily inactivate both HUK and RUK at the lo-’ M level.

Reagents have emerged from these studies which readily distinguish HUK from the urinary plasminogen activator, urokinase. Arginine chloromethyl ketones specifically prepared for the kallikreins exhibit a low level of reactivity with urokinase. For example, in contrast to the high level of reactivity of D-Phe-Phe- ArgCH&l and Dns-Pro-Phe-ArgCH&l with the urinary kallikreins, inhibition of urokinase occurs only at concentrations 3


orders greater in magnitude (unpublished observation). On the other hand, urokinase is readily inactivated by Glu-Gly-ArgCH&l and Ac-Gly-Gly-ArgCH,Cl at the 10e6 to lo-’ M levels while HUK reacts with these reagents only at concentrations 3 to 4 orders greater in magnitude (18). A reagent has not been obtained which will distinguish HUK from plasma proteases. The reactivity of Pro-Phe-ArgCH,Cl and Ala-Phe- ArgCH,Cl with human plasma kallikrein, recently reported (14), is 300- and lOOO- fold greater, respectively, than that of HUK. In fact, almost all peptides of arginine chloromethyl ketone are more reactive with plasma kallikrein than HUK. Although both proteases exhibit a preference for reagents with Phe in the P, position, there are large differences between the relative reactivities of the plasma and urinary kallikreins with varia- tion in the P1, PZ, PB, and P, residues yielding unique patterns of reactivity for each protease. When necessary to distinguish plasma from urinary kallikrein in viva, where less quantitative physiological assays are used, this pattern of reactivities should make it possible to identify either enzyme. For example, the ratios of reactivities of reagents XXIV, XIV, XV, and II with HUK (Table II) are 1.0/85/34/500 while for plasma kallikrein the ratios are 1.0/1.2/0.01/9.1.

A considerable amount of information has been obtained on the specificity of HUK for the -Arg-Ser- bond in the -Gly- Phe-Ser-Pro-Phe-Arg-Ser- sequence of kininogen hydrolyzed by HUK in the liberation of the C terminal of Lys- bradykinin. Comparison of the affinity of HUK for Ac-Phe-ArgCH,Cl, Pro-Phe- ArgCH,Cl, Ac-Pro-Phe-ArgCH,Cl, Ser- Pro-Phe-ArgCH,Cl, and Phe-Ser-Pro- Phe-ArgCH,Cl revealed that values of the Ki for the first two reagents were almost identical and that the Ki for the pentapeptide analog was twofold lower. These results suggest that Arg in the P, position and Phe in the P, position and P, position are important in determining the specificity of HUK for its physiological substrate. On the other hand, HUK had

three- to fourfold lower affinity for Ac- Pro-Phe-ArgCH,Cl and Ser-Pro-Phe- ArgCH,Cl than for Ac-Phe-ArgCH,Cl indicating that Pro and Ser in the P3 and P, binding sites are either of limited or no importance. The lower affinity of the acetylated tripeptide and tetrapeptide analogs maybe due to the loss of a H-bonding site of the reagent due to acylation of the P3 proline if H bonding in the subsites of HUK is homologous to that of chymotrypsin (27) and subtilisin (28). The differences we have observed between the reactivity of substrate analogs with HUK may be due in part to differences in the conformation of reagents in solution.

Comparison of the reactivity of arginine chloromethyl ketones with Lys and homoarginine analogs revealed in each case that HUK was lo-fold more reactive with the arginine chloromethyl ketone. The preference of HUK for Arg over Lys in the P, position is considerably smaller than that of plasma kallikrein (14) and comparable to that of trypsin (17). The small difference between the reactivities of the arginine and homoarginine chloromethyl ketone appears to be unique for HUK since for other proteases examined a larger difference was observed, usually 2 to 3 orders in magnitude (unpublished observation). Substitution of Gly for Phe in the P, position of Pro-Phe-ArgCH,Cl decreased the reactivity of the reagent 150-fold thus confirming the importance of Phe in this position. The replacement of the P, Pro of Pro-Phe-ArgCH,Cl by Phe, Ala, or Glu had no significant effect on reagent effectiveness indicating a limited role of binding of the P3 position in determining substrate specificity.

Examination of the reactivity of RUK with the arginine chloromethyl ketones revealed that, like HUK, it is more reactive with reagents which correspond to the amino acid sequence of kininogen than other reagents. However, the affinity of RUK for these reagents is approximately lo-fold as great as that of HUK. Furthermore, Arg in the P, position, Phe in the P, position and to a lesser extent Pro in the P, position, appear to be of

428 KETTNER ET AL.

greater importance than for HUK in determining the specificity of RUK. Sub- stitution of Lys and homoarginine for Arg in the P, position decreased the reactivity of RUK with the reagent 200- and 1500-fold, respectively, and substitution of Gly for Phe in the P, position decreased reactivity by a factor of 250. Finally, substitution of Glu, Ala, and Phe for the P, Pro decreased the reactivity of RUK by factors of 3 to 4.

Comparison of reactivities of HUK and RUK with the chloromethyl ketones indicated that they are similar in many respects, but distinctive differences exist between them. The most notable differences are the greater preference of RUK for the P,Arg and the greater reactivity of RUK with arginine chloromethyl ketones in general. On the other hand, similarities between HUK and RUK are clearly shown by their parallel reactivities (Table II). For example, reagents containing Phe in the P, position were the most effective inhibitors for both enzymes; reagents with Val and Pro in the P, position were intermediate in effectiveness and reagents with Gly in the P, position were least effective. For each protease, substitution of D-Phe for Phe significantly increased the reactivity of the affinity label. A similar preference for D-Phe in the P, position has been observed for trypsin and a number of other trypsin-like enzymes (unpublished observation). Homology among these enzymes may be reflected in this phenomena.

The differences we have observed in the responses of HUK and RUK to arginine chloromethyl ketones, although small in many cases, are in contrast to our observation with human and bovine thrombins in which the limited number of reagents tested with thrombin from both species were identical in reactivity (15, 16).

In conclusion, we have prepared a number of reagents which are effective in the inactivation of human and rat urinary kallikreins. These reagents should be beneficial in the clarification of the physiological role of glandular kallikreins. Ap- plication of these reagents to the investiga-

tion of a glandular kallikrein has already been demonstrated with the use of D-Phe- Phe-ArgCH,Cl to inactivate a protease in the bladder of toads (Bufo marinus) shown by the inhibition of sodium transport (29). This kininogenase appears to be analogous to renal kallikrein associated with the distal nephron of the mammalian kidney (30).

ACKNOWLEDGMENTS

The authors would like to extend their gratitude to F. C. Brown for her excellent technical assistance, to Dr. J. Iwai and M. A. Heine, Medical Depart- ment, Brookhaven National Laboratory, for assistance in conducting bioassays, to Dr. K. Shimamoto and Dr. H. S. Margolius, Department of Pharmacology, Medical University of South Carolina, Charleston, for determining kininogenase activity in vitro, and to Dr. J. Chao also of the Medical University of South Carolina for making rat urinary kallikrein available to us.

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KETTNER ET AL.

active site mapping of human and rat urinary kallikreins by peptidyl chloromethyl ketones

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