Specificity and inhibition studies of human renal dipeptidase

Biochimica et Biophysica Acta, 956 (1988) 110-! 18 Elsevier

110

BBA 33197

Specificity and inhibition studies of human renal dipeptidase Benedict J. Campbell, Yuan Di Shih, Lawrence J. Forrester and Warren L. Zahler Department of Biochemistry, University of Missouri, Cohimbia, MO (U.S.A.)

(Received 8 April 1988) Key words: Renal dipeptidase: Peptidase specificity: Inhibition kinetics: Leukotriene D4; (Human)

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of V,,,a,,/K,, , for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substratebinding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordk ation site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transiti~,-.tate peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that ~statin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the/Mactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene 1)4 to leukotriene E 4 in the presence of human renal ¢:~m0"~tidasew~s demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatazing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay. The specific activity of the enzyme at 0.1 mM leukotriene D4 was 12.1/zmol ieukotriene D4 per min per mg enzyme.

Abbreviations: LTC4, leukotriene C4; LTD4, leukotriene 134: LTE 4, ieukotriene E4: Mops, 4-morpholinepropanesulfonic acid: TEA, triethylamine; PITC, phenylisothiocyanate. Correspondence: B.J. Campbell, Department of Biochemistry, University of Missouri, Columbia, MO 65212, U.S.A.

Introduction Human renal dipeptidase (dipeptide hydrolase, EC 3.4.13.11) has been purified from human kidney cortex and has been shown to catalyze the hydrolysis of the unsaturated dipeptide, glycylde-

0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

111

hydrophenylalanine, as well as the fl-lactam antibiotic, imipenem [1,2]. The enzyme has been shown to be membrane-bound [2] and could be characterized as a microsomal dipeptidase. The dipeptidases from hog kidney [3] and rat kidney [4] have been shown to convert leukotriene D4 to leukotriene E4, and our preliminary results have indicated that the human enzyme also catalyzes this reaction [5]. In the present paper the human enzyme is distinguished from other mammalian peptidases on the basis of substrate specificity, and the effect of the amino acid sequence of the dipeptide substrate upon the rate of enzyme-catalyzed hydrolysis is studied. The inhibitory effects of the chelating agent, dithiothreitol, and the transition-state peptide analog, bestatin, upon the kinetics of dipeptide hydrolysis are shown to differ in mechanism. Also, the fl-lactam inhibitor, cilastatin, is employed as a kinetic probe to demonstrate whether the subunits of the oligomeric enzyme react independently during the catalytic event, or whether they interact to produce a cooperative effect. The leukotrienes C4, D4 and E4 are known collectively as the sulfidopeptide leukotrienes, and are agents that exhibit potent actions on smooth muscle. In the work reported here, it is shown that purified human renal dipeptidase catalyzes the hydrolysis of the cysteinylglycine bond in leukotriene D4 to convert leukotriene I)4 to leukotriene E4. It has been previously suggested that endogenously produced sulfidopeptide leukotrienes could play a role in the regulation of renal hemodynamics and could modify urinary electrolyte excretion [6]. Materials and Methods

Purification of human renal dipeptidase The enzyme was purified from human kidney cortex using procedures previously described [2]. The activity against glycyldehydrophenylalanine was concentrated from kidney by homogenization, 1-butanol solubilization and (NH4)2SO4 fractionation. Final purification was achieved by highpressure liquid chromatography (HPLC) followed by affinity chromatography.

Enzyme assays The activity of the purified renal dipeptidase was determined by measuring the rate of enzymecatalyzed hydrolysis of the unsaturated dipeptide, glycyldehydrophenylalanine [7]. The fall in absorbance of a solution of 5.00.10 -5 M of the peptide in Tris-HCl buffer (pH 7.6) was measured at 275 nm (1-cm light path). Enzyme units are expressed as micromoles of substrate hydrolyzed per rain, and specific activity is expressed as micromoles of substrate hydrolyzed per min per mg enzyme. Protein concentrations were determined by the method of Lowry et al. [8]. The peptidase-catalyzed hydrolysis of saturated dipeptides was followed by adapting an analytical method for amino acids based upon pre-column derivatization of the amino acids with phenylisothiocyanate followed by HPLC [9l. In a typical assay the enzyme was added to a mixture of 100 /,tM dipeptide, 18/.tM histidine (internal standard), 12 mM Mops buffer at pH 7.1 to give a final volume of 50 ttl. The relationship between purified enzyme concentration and enzyme activity against LTD 4 was established over the range of 1-69 ng enzyme in the reaction mixture. The reaction mixture was incubated at 37°C, and 10 #1 aliquots were removed at appropriate time intervals from 30 s to 15 rain. The reaction was stopped by addition of the aliquots to 90 #1 of M e O H / T E A / P I T C (119 : 15 : 1), and derivatization of reaction products was carried out by maintaining these mixtures at room temperature for 20 rain. The product mixtures were dried under vacuum for 1½ h and stored over - 1 0 °C prior to HPLC analysis. Solvent A employed for HPLC separation was 0.14 M sodium acetate containing 0.5 ml/liter TEA adjusted with concentrated acetic acid to pH 6.35, and solvent B was 70% acetonitrile and 30% water. Both solvents A and B were filtered and degassed prior to use .The derivatized residues were dissolved in 100/,tl of solvent A, and 50/~1 of this solution was injected on to a Econosphere C!8 column (150 ×4.6 ram) which had been equilibrated with a mixture of 95% solvent A and 5% solvent B. The products were eluted from the column by changing the eluant from 5% solvent B to 30% solvent B over a period of 7 rain. The column was cleared by changing the eluant from 30% solvent B to 100% solvent B over a period of

112

10 min. The flow rate was 1 ml/min, and absorbance was recorded at 254 nm. The HPLC analysis was calibrated by using solutions containing known amounts of glycine and histidine. A standard curve was constructed by plotting the ratio of the height of the glycine peak to the height of the histidine peak vs. picomoles glycine present in the control solutions. This standard curve was linear over the range of 5-600 pmol glycine. The amount of glycine released during the course of peptide hydrolysis was then plotted as a function of time, and the velocity of the enzyme-catalyzed reaction was calculated from the resulting linear plot. Aliqouts from a control reaction mixture without enzyme were analyzed at various times to determine the stability of the peptide under these conditions. All HPLC experiments were performed using a Beckman model 110A HPLC chromatograph with a Hitachi model 100-10 spectrometer and a Fisher Recordall, series 5000. Other peptidase assays were employed as follows: Aminopeptidase A (L-a-aspartyl(L-a-glutamyl)-peptide hydrolase, EC 3.4.11.7) was assayed using L-aspartic acid-fl-naphthylamide as substrate using the method reported by Lalu et al. [101. Aminopeptidase B (L-arginyl(L-lysyl)-peptide hydrolase, EC 3.4.11.6) was assayed using L-arginine-fl-naphthylamide according to the procedure described by Kawata et al. [11]. The assay for aminopeptidase M (a-aminoacyl-pepfide hydrolase (microsomal), EC 3.4.11.2) [12] was carried out at 37°C in a reaction mixture which coptained either 1.7 mM [Leu]-enkephalin or 1.5 mM of [Met]-enkephalin and 0.4 #g of purified human dipeptidase in 50 mM Mops buffer at pH 7.1. The total volume was 32 #i from which 15 #! aliquots were taken after 2 h incubation. Commercial aminopeptidase M (Sigma Chemical Co.) was substituted for human dipeptidase as a positive control. Th~ aliquots were analyzed by cellulose thin-layer chromatography using methanol/ water/pyridine (80 : 20 : 4) as the developing solvent. The chromatograms were sprayed with 0,4% ninhydrin in ethanol for color development. Aminopeptidase P (aminoacylprolyl-peptide hydrolase, EC 3.4.11.9) [13] and tripeptidase (aaminoacyl-dipeptide hydrolase, EC 3.4.11.4) were assayed by monitoring the release of amino acids as a function of time from their respective sub-

strates, glycyl-L-prolyl-L-alanine and L-phenylalanylglycylglycine. In th~,se assays the procedure of pre-column derivatization of amino acid products followed by HPLC as described above was employed. Materials

Leukotriene D4 was kindly provided by Dr. J. Rokach of Merck-Frosst, Pointe Claire, Canada. Dipeptides, tripeptides, enkephalins, L-aspartic acid-fl-naphthylamide and L-arginine-fl-naphthylamide were purchased from Sigma Chemical Co., St. Louis, MO. Cilastatin and imipenem were gifts from Dr. H. Kropp, Merck, Sharp and Dohme. Bestatin hydrochloride was supplied by Sigma Chemical Co., St. Louis, MO. Dithiothreitol was obtained from Schwarz/Mann Biotech, Cleveland, OH. Phenylisothiocyanate, pyridine and triethylamine were purchased from Pierce Chemical Co., Rockford, IL. The Econosphere C!8 HPLC column was purchased from Allteck Associates, Deerfield, IL. GLycyldehydrophenylalanine was synthesized by methods previously described [14]. Results and Discussion

The human dipeptidase, purified by previously described methods [2], v~as shown to be homogeneous by analytical polyacrylamide gel electrophoresis and HPLC. The specific activity of the enzyme was determined to be 128.2 #mol substrate hydrolyzed per min per mg protein, using the standard glycyldehydrophenylalanine assay at a substrate concentration of 5.00.10 -5 M. To show that the human dipeptidase is a unique enzyme with properties that differ from other similar peptidases, the specificity of the enzyme was measured against that are characteristic for other known mammalian peptidases. Aminopeptidase A (substrate: L-aspartic acid-fl-naphthylamide) catalyzes the hydrolysis of peptide bonds in which an acidic amino acid contributes the carbonyl function, and aminopeptidase B (substrate: L-arginine-fl-naphthylamide) catalyzes the hydrolysis of a peptide bond in which a basic amino acid contributes the carbonyl function. Aminopeptidase M (substrates: [leu]-enkephalin and [met]-enkephalin) catalyzes the hydrolysis of

113 TABLE I KINETIC PARAMETERS OF HUMAN RENAL PEPTIDASE AGAINST GLYCYL DIPEPTIDES Dipeptide

Km a (mM)

//max (#mol/min per mg)

L-Leu-Gly Gly-L-Leu

0.21 1.22

67 234

312 193

L-Phe-Gly Gly4-Phe

0.67 0.99

321 314

481 317

L-Trp-Gly Gly-L-Trp

0.15 2.03

55 231

367 114

L-VaI-Giy Gly-L-Vai

0.09 11.0

108 15

1,250 1.3

the second position. Finally tripeptidase (substrate: L-Phe-Gly-Gly) employs tripeptides as substrates. The purified human renal dipeptidase exhibited no detectable activity against any of these substrates. When the activity of the dipeptidase was tested against a series of dipeptides, it was observed that enzyme-catalyzed hydrolysis of the dipeptide bonds occurred at different rates Pairs of dipeptide substrates were selected such that glycine was positioned at either the C-terminal or the N-terminal residue. The rates of glycine release by purified dipeptidase was then measured using the phenylisothiocarbamoyl derivative-HPLC assay described above. The kinetic parameters, Kn, and Vma~, were determined from Lineweaver-Burk plots [15], and the effect of dipeptide sequence on the kinetic parameters is reported in Table I. The ratio of Vmax/K m is used as an index of the enzyme's preference for substrates. According to the data presented in Table I. the enzyme prefers that the bulky hydrophobic group of the dipeptide be located at the N-terminal position. Also, the substrate of each dipeptide pair that exhibits the lower K m is the dipeptide with the bulky group in the N-terminal position. This suggests that the

DI-

Vmax/K m

'~ All rate measurements were made by the pre-column derivatization-HPLC method described in the Materials and Methods. The kinetic parameters were determined using the standard graphical procedures of Lineweaver and Burk [15].

the N-terminal amino acids from polypeptide chains. Aminopeptidase P (substrate: glycyl-L-prolyl-L-alanine) hydrolyzes the peptide bond between the first and the second residues at the N-terminus of a peptide chain if proline occupies 0.100.090.080.07

•

•

•

g

0.06

1 V (pmole/min/mg) "1 m

0.05 0.04 0.03. 0.02. 0.01, ,r

i

-30.0

!

-20.0

-10.0

0

2 .0

3 .0

!

40.0

!

50.0

60.0

(S) (raM) Fig. 1. Double-reciprocal plots of dithiothreitol-induced inhibition of human renal dipeptidase. The dipeptidase-catalyzed rates were measured against glycyldehydrophenylalanineat pH 7.1 and 37 ° C. The substrate concentration was varied over the range 0.02 to 0.13 mM. Rates are shown in both the presence and the absence of the inhibitor, dithiothreitol. Inhibitor concentrations are 0.15 and 0.30 mM.

114

this system. The mechanism of inhibition appears to be a competition of the inhibitor with substrate for a position within the active site of the enzyme. It seems plausible that the group involved is a coordination site of zinc which would be expected to complex readily with one or more of the thiol groups of the inhibitor, dithiothreitol. The transition-state peptide analog, bestatin, has been shown to inhibit aminopeptidase B [19] and aminopeptidase M [20] as well as other membrane-bound aminopeptidases, but it has been reported not to inhibit rat renal dipeptidase [4,21]. To determine the effect of bestatin upon purified human dipeptidase, the rate of dipeptidase-catalyzed hydrolysis of glycycldehydrophenylalanine was measured at various substrate concentrations in the presence and absence of the peptide analog. The initial experiments indicated that the bestatin-induced inhibition was time dependent and that an initial preincubation period of enzyme with inhibitor for 10 rain was necessary to estab-

substrate binding site of the enzyme may provide a hydrophobic pocket for positioning the substrate within the active site. Agents that have been shown to regulate the rates of peptidase-catalyzed reactions include metal-chelating compounds [16], transition-state analogs [17], and, in certain cases, fl-lactam reagents [2]. A kinetic analysis of the inhibition of the purified peptidase by dithiothreitol is presented in Fig. 1. The standard graphical technique of Lineweaver-Burk [15] was employed. The data points are the average experimental values for three independent rate measurements. The lines ar,~ drawn by computer calculation using the equation for linear competitive inhibition [18]. The kinetic parameters for the dipeptidase using glycyldehydrophenylalanine as substrate are Vmax ffi 141.2 :t: 5.1 /zmol/min per rag, Km= 0.059 + 0.005 mM, and the inhibition constant for dithiothreitol is K i = 0.098 + 0.006 mM. The data demonstrate reversible, competitive inhibition for

O.OS

1

0.04 .]

0.03

1 V(pmolee/mln/mg) 0.02

0,01

-30.0-20.0-10.0

0

10.0

20.0

80.0

40.0

50.0

60.0

70.0

80.0

1 $(mM) Fig. 2. Double-reciprocal plots of bestatin-induced inhibition of human renal dipeptidase. The dipeptidase-catalyzed rates were measured against glycyidehydrophenylalanine at pH 7.1 and 37 o C. Enzyme was pre-incubated with inhibitor for 10 rain prior to addition of substrate. The substrate concentration was varied over the range 0.01 to 0.13 raM. Rates are shown in both the presence and the absence of the inhibitor, bestatin. Inhibitor concentrations are 2.9 and 5.8/L M.

115 1,0"

x

x

0.9. 0.8. 0.7

0.6

V

0.5. 0.4' 0.8' 0.2' x

0.1 0

o oo5

o.~,1

o.~)5

o11

o~5

K

K

11o

210

(CilastaUrl)(pM) Fig. 3. Cilastatin-induced inhibition of human renal dipeptidase. The velocity of tile reaction was measured in the presence and absence of cilastatin by the spectrophotometric assay using glycyldehydrophenylalanine as substrate at pH 7.1 and 37 o C. The substrate concentration was 0.04 mM, and the cilastatin concentration varied from 0.001 to 2.0 #M. In the equation, Y is the fractional velocity, [I] is the inhibitor concentration, and 150 is the inhibitor concentration that produces 50% inhibition. The line ( ~ ) is calculated from Eqn. 1 and the symbols ( × ) represent data points.

Optical Density 280nm 0.00,4 -

0.003

-

0.002

-

LTD4

LTE4

o oo

LTD4 d

0

i [~

|

4.0

N .o

1 rain R e a c t i o n

Time

LTE4 f

~,,,,, ~,~~,, 0 4.0 6.0 8.0 10.0 12.0 14.0

5 min Reaction

Time (rain)

Time

Fig. 4. Conversion of LTD 4 to LTE4 catalyTed by human renal dipeptidase. Reaction mixtures containing 5.0 #M LTD4 and 10 mM Mops buffer at pH 7.1 in a final volume of 55 #! were preincubated at 3 7 ° C for 5 rain. The reactions were initiated by the additi~,n of 58 ng of dipeptidase in 10/~! Mops at pH 7.1. The reactions were terminated at various times by the addition of 55 #1 of 0.5% acetic acid in methanol, and 50/~1 aliquots were removed for analysis by HPLC on a CI8 reverse-phase column. The column was equilibrated and eluted with methanol/water/acetic acid (700:300:1) which had been adjusted to pH 5.4 with NH4OH. The flow rate was 1 ml/min, and absorbance was recorded at 280 nm.

116

lish a constant velocity over the period the rate of reaction was measured. The kinetic data obtained were analyzed by the standard graphical technique of Lineweaver-Burk as described above. The results shown in Fig. 2 demonstrate bestatin-induced apparent non-competitive inhibition of the enzyme. The calculated inhibition constant for bestatin is 4.25 +0.18 #M. The results of the inhibition studies presented in Figs. 1 and 2 consistantly demonstrated competitive inhibition by dithiothreitol and non-competitive inhibition by bestatin. When the kinetic studies were performed with different enzyme preparations five different times, a range of K m values from 0.040-0.060 mM (mean 0.053) were obtained. In the absence of pre-incubation, bestatin produces a time-dependent decrease in reaction rate which reaches steady state after 10 rain. Once steady state is reached, classical non-competitive inhibition is seen, where the Km of glycyldehydrophenylalanine is essentially independent of inhibitor concentration. The most likely explanation of these results is that bestatin initially forms a weak complex with enzyme, which slowly undergoes a conformational change to a tight-binding complex. Previous results reported from our laboratory indicated that the human dipeptidase has a molecular weight of 220000, estimated from appropriate protein standards measured by analytical HPLC [2], Dissociation of the enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a single polypeptide (M r 59000). Analysis of the peptidase for zinc content gave 3.9 g atoms of zinc per reel of enzyme. These results indicated that the native enzyme contains four subunits of Mr 59000 [2]. It is possible to determine whether the subunits of an oligomeric enzyme react independently during the catalytic effect, or whether they interact to produce a cooperative effect. Generally, identical subunits react independently. That is, inhibition of an oligomeric enzyme with identical and noncooperative subunits would be kinetically indistinguishable from a monomeric enzyme. The velocity produced by such an oligomeric enzyme would depend o n substrate concentration according to the Michaelis-Menten equation for competitive inhibition. If this equation is divided by the form of the equation with no inhibitor present, a

relationship between fractional velocity and inhibitor concentration is obtained [22]. These expressions are shown below. v[s] f K~/1 + + IS] Ki !

v=

v --

=

Y =

Vo

(1)

15o /so + [ll

~

(2)

The upper expression relates the velocity of the enzyme-catalyzed reaction to substrate concentration in the presence of a reversible, competitive inhibitor. The lower expression results when the upper is divided by the same expression without inhibition present. The quantities in the lower expression are Y, the fractional velocity; 15o, the inhibitor concentration which causes 50~ inhibition; and [I] the inhibitor concentration. Applying this equation, a plot of Y vs. Ill gives a symmetrical graph with a point of inflection at [I] equal to is0 where the slope is maximal. A theoretical curve can be constructed using values of Y from 1 to 0.1 to calculate corresponding values of [1]. Normally, the inhibitor concentration range should span a

2.5-

i 2.0 e

g 1.o.

0.5-

,._........._._._....

.....

---,--O

Time (rain) Fig. 5. Human renal dipeptidase-catalyzed release of glycine from LTD 4 as a function of time. The reaction was followed by pre-column derivatization of glycine with phenylisothiocyanate followed by HPLC as described in Materials and Methods. The enzyme-catalyzed reaction is represented by the linear regression line ( ~.), and the control without enzyme present is represented by ( . . . . . . ).

117

more than 100-fold range, and it becomes more convenient to plot log[l]. The log[I] equals log 150 at the inflection point. In our previous studies, we had established that cilastatin was a reversible, competitive inhibitor of the human renal dipeptidase [2], and we selected cilastatin as the inhibitor to be employed in these studies. A comparison of the experimental data with the calculated theoretical curve is shown in Fig. 3. The value of 150 calculated from the inflection point of the curve is 0.08/~M. The fit of the data points to the theoretical curve indicates that the subunits of the oligomeric enzyme react independently during enzyme catalysis and that the rate of the reaction is uot influenced by cooperativity between sites on the subunits. Since there are four subunits of the same

size with four gram atoms of zinc per ~etramer, and no cooperativity between subunits, it seems plausible to suggest that the subunits are identical. Final confirmation of the presence of chemically identical subunits must await the determination of their sequence. The conversion of LTD 4 to LTE4 by enzymecatalyzed hydrolysis is shown in Fig. 4. During incubation of LTD4 with purified dipeptidase, aliquots were removed at appropriate times, and the products were isolated by HPLC. The breakdown of LTD 4 is demonstrated as the area under the LTD 4 peak is diminished and the LTE 4 peak is increased with time. The rate of this reaction was obtained by measuring the release of glycine from LTD 4 as a function of time as described in Materials and Methods. LTD,-DipopiidasoActivity moles LTO,/ein/el

Dil)OlslidaseAclivil!!

nmolasGiP/min/ml

I-I00

)(xx

90.0 -,

0

80.0 4

I

r9.0

~

F8.0

70.0 4

7

~

~-7.0

60.0 4

p

~

t-6.o

50.04

I

40.0 4

I

30.0 4

I

~-5.0

o !

F4.0

I

20.0 4

I

!

~o.o -i

/

OL

o

~.3.o ~

t-2.0

\

F 1.0 \

95

100

0 105

110

115

120

EiuUon Volume (ml) Fig. 6. Analytical HPLC rechromatography of purified renal dipeptidase assayed against ieukotriene D 4 and glycyldehydrophenylalanine. The dipeptidase was rechromatographed by reverse-phase HPLC using a Toya Soda TSK 3000 column equilibrated with 50 mM Tris-HC! buffer at pH 7.1. The flow rate was 4 ml/min, and 2 ml fractions were collected. All fractions were analyzed using leukotriene D 4 (0.08 mM) and glycyldehydrophenylalanine (0.05 mM) as substrates as described in Materials and Methods.

118

The time-course of the reaction is shown in Fig. 5. The regression line drawn through the experimental data points demonstrates a linear relationship between glycine released and time, with a correlation factor of 0.994. The plot obtained with no enzyme present shows that no significant breakdown of LTD 4 occurs under the conditions of the assay. The relationship between the purified enzyme concentration and enzyme activity against LTD 4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay. The specific activity of the enzyme at 0.1 mM LTD under the reported conditions was 12.1 /~mol LTD 4 hydrolyzed per rain per mg enzyme. To confirm that the same enzyme acted upon both glycyldehydrophenylalanine and leukotriene D 4, the purified human dipeptidase was rechromatographed by HPLC as reported in Fig. 6. The eluted fractions were assayed using glycyldehydrophenylalanine and LTD 4 as substrates in individual assays. The enzyme activity was eluted as a single symmetrical peak with activity against glycyldehydrophenylalanine congruent with the activity against LTD4. Activity was not detected in any other fractions of the elution pattern. Although the data show conclusively that human renal dipeptidase, catalyzes the hydrolysis of LTD 4, the enzyme is not specific for LTD 4, and the physiological significance of this enzyme in the bioconversion of LTD 4 to LTE4 is not certain. Acknowledgements This work was supported in part by a grant from University of Missouri Medical School Research Council (DHHS BRS 5387), and it is a contribution from the Missouri Agricultural Experiment Station. Journal Series No. 10390.

References I Kropp, H., Sundelof, J.G., Hajdu, R. and Kahan, F.M. (1982) Antimicrob. Agents Chemother. 22, 62-70. 2 Campbell, B.J., Forrester, L.J., Zahler, W.L. and Burks, M. (1984) J. Biol. Chem. 259, 1486-14590. 3 Farrell, C.A., Allegretto, N.J. and Hitchcock, M.J.M. (1987) Arch. Biochem. Biophys. 256, 252-259. 4 Kozak, E.M. and Tate, S.S. (1982) J. Biol. Chem. 257, 6322-6327. 5 Shih, I., Forrester, L.J., Zahler, W.L. and Campbell, B.J. (198~ Fed. Proc. 45, 1193. 6 Filep, J., Rigter, B. and Frolich, J.C. (1985) Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18), F739-F744. 7 Rene, A.M. and Campbell, B.J. (1969) J. Biol. Chem. 244, 1445-1450. 8 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 9 Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1984) J. Chromatogr. 336, 93-104. 10 Lalu, K., Lampelo, S., Nammelin-Kortelainen, M and Vanha-Perttula, T. (1984) Biochim. Biophys. Acta 789, 324-333. 11 Kawata, S., Takayama, S., Ninomiya, K. and Makisumi, S. (1980) J. Biochem. 88, 1025-1032. 12 Chaiilet, P., Marcais-Collado, H., Costentin, J., Yi, C.C., Baume, S.D.L. and Schwartz, J.C. (1983) Eur. J. Pharmacol. 86, 329-336. 13 Dehm, P. and Nordwig, A. (1970) Eur. J. Biochem. 17, 364-371. 14 Campbell, B.J., Lin, Y. and Bird, M.E. (1963) J. Biol. Chem. 238, 3632-3639. 15 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658-666. 16 Hooper, N.M., Low, M.G. and Turner, A.J. (1987) Biochem. J. 244, 465-469. 17 Shenvi, A.B. (1986) Biochemistry 25, 1292-1299. 18 Cleland, W.W. (1979) Methods Enzymol. 63, 103-138. 19 Umezawa, H., Aoyagi, T., Suda H., Hamada, M. and Takeuchi, T. (1976) J. Antibiot. 29, 97-99. 20 Suda, H., Aoyagi, T., Takeuchi, T. and Umezawa, H. (1976) Arch. Biochem. Biophys. 177, 196-200. 21 Mclntrye, T. and Curthoys, N.P. (1982) J. Biol. Chem. 257, 11915-11921. 22 Tahir, M.K. and Mannervik, B. (1986) J. Biol. Chem. 261, 1048-1051.