Biochemical characterization and substrate specificity of rat prostate kallikrein (S3): comparison...

Biochimica et Biophysica Acta. 1121 (1992) 309-316 309 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34226

Biochemical characterization and substrate specificity of rat prostate kallikrein ($3): comparison with tissue kallikrein, tonin

and T-kininogenase

Cindy Wang, Caroline Q. Tang, Gary X. Zhou, Lee Chao and Julie Chao Department of Bitn-ht, mist~" and Molecular Biology Medical Unie'ersiO' of South Carolina. Charleston. SC (USA)

(Received 26 September 1991)

Key words: Prmtate: Kallikrein; Tonin; Kininogenase

A tissue kallikrcin-likc cnzymc encodcd by $3 mRNA was purified to homogencity from rat prostate gland. Thc apparent molecular mass of the prostate enzyme is 32 kDa as detc: 'mincd by so,Jium dodccyi sulphate polyacrylamidc gel clcctrophoresis (SDS-PAGE). The intact 32 kDa enzyme is split into two bands of lower molecular mass, 18 and 14 kDa, under reducing conditions on SDS-PAGE. NH,-terminal amino acid scqucnce a,alyses ,of the intact enzyme and heavy and light chains revealed the idcr~.tity to the translated ~quence of a prostate kallikrein eDNA ($3). lsoelectric focusing indicated that the prostate enzyme is a basic protein with p l of 7.30-7.45. Specific activities of the prostate kallikrcin toward angiotensin I, angiotensinogen and rat low M r kininogen as well as tr ipeptide chromogenic substrates were compared with thosc of tissue kallikrein, tonin and T-kininogenase. The kinin-releasing activity is inhibited by lcupeptin, antipain, benzamidine and soybean trypsin inhibitor. A sensitive and specific radioimmunoassay fur the rat prostate kallikrein shows that thc immunoreactive kallikrein Icvcls in prostate and submandibular gland were 23.78 + 2.62 /zg/mg protein (n = 5) and 12.29 + 2.25/,tg/mg protcin (n = 5), respectively. The results indicate that the prostate kallikrcin $3 is cxpresscd at high levels in both prostate and submandibular glands.

Introduction

Tissue kallikreins (E.C 3.4.21.35) are a subgroup of serine proteinases that are encoded by multigene fami- lies in rat, mouse and possibly human [1-4]. Kallikrein multigene family enzymes are involved in the post- translational processing of polypeptide hormone and growth factor precursors to their biologically active forms [5]. The best characterized biological function of kallikreins is their ability to release vasoactive kinins from kininogen precusor~. In vitro, kallikreins have been shown to process a wide variety of peptide precursors including prorenin, proinsulin, atrial natriuretic

Abbreviations: TEMED, N,N,N'.N'-tctramethylethylenediamine; DTT, dithiothreitol; low M r kininogen, low-molecular weight kininogen; PMSF, phenylmethylsulfonyl fluoride; RIA, radioimmunoassay; pNa, p-nitroanilide; MCA, methylcoumarin amide; Cm-papain, car- hoxymethyl-papain.

Correspondence: J. Chao, Department of Biochemistry and Molecu- lar Biology, Medical University of South Carolina. 171 Ashley Av- enue. Charleston, SC 29425. USA.

factor, apolipoprotein B 100, plasminogen and angio- tcnsinogen. The large family of kallikrein-likc genes has been implicated in a plethora of physiological functions.

The diversity of kallikrein functions depends on tissue-specific expression patterns, substrate specificity and hormonal regulation [6]. Localization of kallikrein- like enzymes and their mRNAs has been demonstrated in variou~ exocrine and endocrine organs including kidney, salivary gland, par~creas, brain, pituitary, skele- tal muscle, heart, vasculature and prostate gland. Tis- sue kallikrcins, due to their ability to release vasoactive kinins from kininogen precursors, are known to medi- ate changes in local blood flow and ion transport [7]. Furthermore, other kallikrein family members have defined roles as well. For example, the y-subunit of nerve growth factor is involved in processing nerve growth factor in the mouse [8]; epidermal growth factor-binding proteins A, B and C process mouse epidermal growth factor [9], T-kininogenase cleaves T- kininogen to release T-kinin in the rat [10,11]; a kallikrein-likc enzyme from rat submandibular gland has growth-promoter activity for the proliferation of adipose precursor cells [12] and finally, studies showed

310

that an arginine esterase encoded by $3 mRNA from rat submandibular gland extract has potent vasocon- strictor activity [13]. The apparent substrate specificity of individual kallikreins suggests that kallikrein gene family members function to process polypeptide precursors and to play roles in a wide spectrum of biological activities.

The possibility that kallikrein-like enzymes in target tissues may play important physiological and patho- physiological roles provides a stimulus for detailed characterization of kallikrein gene family members. In the rat and mouse, almost all of the kailikrein gene family enzymes are expressed in the submandibular gland, and individual members are expressed in one or a few other tissues. Three kallikrein genes, including renal kallikrein (RSKG 7 or K1) [14,15], tissue (pancreatic) kallikrein and a tonin-like gene (RSKG 3), are expressed in rat kidney [14]. MacDonald and his associates have cloned and sequenced two cDNAs, $3 and Pl which encode prostate kallikreins [16]. Recently, we have isolated a kallikrein-related enzyme from rat prostate gland whose NH_,-terminal amino acid sequence matches the derived protein sequence of the prostate kallikrein cDNA $3, and in the present com- munication we report the purification, characterization and tissue distribution of rat prostate kallikrein $3 as well as comparison of its substrate specificity with tissue kallikrein, tonin and T-kininogena~.

Materials and Methods

Materials The following materials were obtained from com-

mercial sources: DEAE-Sepharose CL-6B (Pharmacia); low-molecular-weight protein standards, bovine serum albumin, 2,2'-dipyridyl, bradykinin, sodium deoxycholate, Coomassie brilliant blue R-250, polybrene, angiotensin i, angiotensin II (Sigma); acrylamide, N,N'-methylenebisacrylamide, TEMED (Eastman Ko- dak), D T r (Aldrich); complete Freund's adjuvant (DIFCO); D-Pro-Phe-Arg-MCA (Enzyme System Prod- ucts), D-Pro-Phe-Arg-pNa, D-VaI-Leu-Arg-pNa, D-lle- Pro-Arg-pNa (Kabi).

Methods Tissue preparation. Rat prostate glands and other

tissues were removed from normal saline perfused Sprague-Dawley rats (Charles River) as described previously [10]. Tissues were trimmed free of fat and connective tissues, immediately minced and then ho- mogenized in 0.25 M sucrose (pH 7.0) containing 1 mM EDTA at 4°C using a polytron homogenizer. The ho- mogenate was centrifuged at 600 x g for 20 min and the supernatant was treated with sodium deoxycholate (0.5%, w/v) for 30 min at room temperature followed by centrifugation at 20000 x g for 90 min. The final supernatant was dialyzed against 20 mM Tris-HCI (pH

8.0) containing 1 mM EDTA. The rat prostate extract was used for further purification.

Enzyme purification. The dialyzed prostate extract was applied to a DEAE-Sepharose CL-6B column (2.2 x 20 cm), pre-equilibrated with dialysis buffer (20 mM Tris-HCl, pH 8.0 containing 1 mM EDTA). After washing, the column was eluted with a 0-0.2 M NaCI linear gradient in dialysis buffer at a flow rate of 35 ml /h . The protein concentration of the eluent was monitored at 280 nm and the activity of the collected fractions were measured by the formation of angiotensin II from angiotensin I and fractions containing angiotensin ll-releasing activity which were determined by an angiotensin il RIA were pooled, dialyzed and lyophylized. The sample was then applied to a HPLC gel filtration column (TSK 250, 7.5 x 300 mm) and the fractions were eluted at 0.3 ml/min with 0.10 M NaCl in 0.01 M sodium phosphate buffer (pH 7.0).

Rat tissue kallikrein [17] from rat submandibular gland, tonin [18] and T-kininogenase [10] from rat submandibular gland were purified to homogeneity.

Angiotensinogen purification. Human angiotensinogen was purified according to the modified method of Hiigenfeldt and Hackenthal [19]. Briefly, human plasma was first fractionated using a 30-45% ammonium sulphate precipitation. The pellets were collected, dialyzed and purified subsequently on DEAE-Sepharose CL-6B and HPLC gel filtration TSK 250 columns. Purified angiotensinogen was resolved on SDS-PAGE and identified as a 55 kDa protein by Coomassie brilliant blue staining. Angiotensinogen content was assayed by angiotensin II generation after incubation with tonin for 30 min at 37°C and the released angiotensin 11 was measured by an angiotensin I1 RIA.

Kininogen purification. Rat low M r kininogen was isolated as described [20]. Rat serum (30 ml) was brought to 10 mM benzamidine, 40/~g/ml polybrene, 2 mM EDTA, 0.2 mM PMSF, 50 /~g/ml soybean trypsin inhibitor and 2 M NaCl. The serum was applied to Cm-papain Affi-Gel 10 column (1.5 × 20 cm) equilibrated with the equilibration buffer containing 50 mM sodium phosphate (pH 7.5), 2 M NaCI, 1 mM benzamidine, 40 /~g/ml polybrene, 1 mM EDTA, 0.2 mM PMSF, 0.02% NaN 3. The column was then washed with equilibration buffer followed by 50 mM sodium phosphate (pH 7.5), 2 mM EDTA, 0.2 mM PMSF and 0.02% NaN 3. Kininogens were eluted with 50 mM sodium phosphate (pH 11.5), 2 mM EDTA, 0.02% NaN 3 in a single peak, which was dialyzed and further purified on an FPLC Mono-Q HR 5 /5 column. Puri- fied rat low M r kininogen with a molecular mass of 68 kDa was identified in SDS-PAGE by Coomassie brilliant blue staining. Rat T-kininogen with a molecular mass of 68 kDa was purified from the serum of rats which were sacrificed 48 h after turpentine treatment [21]. Bovine low M r kininogen was kindly provided by

311

Drs. S. lwanaga and H. Kato of the Protein Research Institute, University of Osaka, Osaka, Japan.

Protein determhmtion. Protein concentration was measured by the Lowry protein assay [22], using bovine serum album;n as the standard. The concentration of purified enzyme was determined spectrophotometri- cally at 205 nm and 280 nm [23].

SDS-polyacrylamide gel electrophoresis. SDS-PAGE was performed as described previously [10] using the buffer system of Laemmli [24].

NH,-terminal sequence analysis. After SDS-PAGE under reducing conditions, the protein was transferred to polyvinylidene difluoride membrane. The protein bands on the membrane were visualized by staining with 0.2% (w/v) Ponceau S in 3% trichloroacetic acid for 1 Imin and destained with 1 M acetic acid for 2 min. The individual bands were cut out and subjected to NH2-terminal sequence analysis using a gas phase protein sequencer equipped with an on-line narrow-bore PTH-amino acid analyzer (ABI model 470A, Applied Biosystems Inc.).

lsoelectric focusing, isoelectrofocusing of the purified enzyme was performed on a 0.5 mm thin layer polyacrylamide slab gel in a pH gradient 3.5-10.0 formed by Ampholine. Separation was carried out for 1 h at constant power (8 watts). Following completion of focusing, the filter paper tabs and edge portion of the gel were removed. Strips (1 cm in width) were cut transversely at 0.5 cm intervals and each section was placed in a tube containing 1 ml of distilled water. After standing about 18 h, the pH values of the sec- tions were determined. The pH gradient was obtained by plotting pH versus gel distance. The remainder of the slab was fixed in 12.5% trichioroacetic acid for 10 min, washed 10 min each, three times with 50% ethanol, 10% acetic acid and finally with water. The protein bands on the gel were visualized with Coomassie brilliant blue staining.

Preparation of antiserum. New Zealand rabbits were injected with 100 ttg of the purified rat prostate kallikrein mixed with complete Freund's adjuvant (DIFCO). Boosts of 100 tLg of the purified enzyme were administered until a high titer of antiserum was detected as described previously [18].

Det, elopment of angiotensin H radioimmunoassay. Angiotensin 11 (5 ttg) was labelled with '251 using a chloramine T method [25]. Antiserum to angiotensin II (kindly provided by Professor Hilgenfeldt at the Uni- versity of Heidelberg, Germany) at a 1:8000 final dilution yields 30% specific binding. 100 ttl of mZSl- angiotensin 11 (10000 cpm/100 ttl) and 100 ~1 of the antiserum were added to 200/~! 1% BSA in PBS for a total vol. of 400 #!. The assay mixtures were incubated at room temperature for 18 h. Antibody-bound angiotensin II w~s separated from free tracer by centrifugation in an optimal combination of 25% poly(ethyl-

ene glycol) (0.8 ml) and 0.1% y-globulin (0.4 ml). The angiotensin II standard curve ranges from 8 pg to 1 ng. The assay showed 3.25% cross-reactivity with angiotensin !.

Det'elopment of prostate kailikrein $3 radioimmunoassay. Rat prostate kallikrein (5 #g) was labeled with ~2-~! by the lactoperoxidase method [26] and the labeled prostate kallikrein was separated from the re- action mixture with a GF-5 column (Pierce Chemical). In the antibody titration curve, rat prostate kallikrein antiserum dilutions in the assay buffer ranged from 1 : 1000 to 1 : 1 000000. 100/.tl of ~ZSl-labeled kallikrein (10000 cpm/100 ill) and 100 g.I of antibody in the assay buffer were added to 200 #.! of assay buffer for a total volume of 400/zl. The assay mixtures were incubated at room temperature for 18 h. Antibody-bound enzyme was separated from free enzyme by centrifugation in an optimal combination of 25% poly(ethylene glycol) and 0.1% y-globulin.

Enzymatic assays using angiotensinogen and kinh~o- gen substrates. Angiotensin ll-releasing activities of prostate kallikrein, tissue kallikrein, tonin and T- kininogenase were measured using angiotensin I or purified human angiotensinogen as the substrate. The reactions were carried out in 50 mM sodium phosphate buffer (oH 7.5) containing 15 mM dipyridyl and 2 mM EDTA. In each tube, 10 ng angiotensin 1 or 10 g.g angiotensinogen was mixed with prostate kallikrein or related enzymes (50 ng) and incubated at 37°C for 30 rain. The reactions were stopped by boiling the sam- ples for 20 min. Aliquots (100 #1) were used to measure the generated angiotensin !I by an angiotensin 11 RIA.

Kinin-releasing activities of prostate kallikrein, tissue kallikrein, tonin and T-kininogenase were measured by incubating the enzyme (50 ng) with rat low M r kininogen, T-kininogen or bovine low M r kininogen (3 /zg) in 0.1 M sodium phosphate (pH 8.5) in a total vol. of 500/zl at 37°C for 30 min. The reactions were also stopped by boiling for 20 min. Released kinin was assayed by a kinin RIA [27].

Initial t'elocity measurements of peptidase actit'ity. The peptidase activities of rat prostate kailikrein, tissue kallikrein, tonin and T-kininogenase were measured by the spectrophotometric assay. Tripeptides o- Pro-Phe-Arg-MCA, D-Pro-Phe-Arg-pNa (S-2302), o- VaI-Leu-Arg-pNa (S-2266) and o-lle-Pro-Arg-pNa (S- 2288) were used as substrates. The molar extinction coefficient for the leaving group 7-amino-4-methylcoumarin (AMC) is 6200 at 370 nm and p-nitroanilide (pNa) is 10500 at 405 nm. The velocity was measured with a Cary 3 spectrophotometer (Varian Associates) using a 0.1 absorbance scale. Reactions were performed at 23°C and initiated by adding varying amounts of each enzyme (10-600 nM) to 0.05 M Tris-HCI (pH 8.0) containing 0.12 mM substrate. Kallikrein-like activ-

312

ity was determined by measuring the linear production of product and the initial velocity is expressed as tool of product formed per min per mol of enzyme.

pH dependence o f rat prostate kallikrein. Rat prostate kallikrein (50 ng) was incubated with rat low M r kininogen (3 #g) in 500 #1 of the three following buffer systems: 0.2 M maleate pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5; 0.2 M Tris-HCI pH 7.5, 8.0, 8.5, 9.0; or 0.2 M glycine-NaOH pH 9.0, 9.5, 10.0, 10.5 at 37°C for 30 min. The reactions were stopped by boiling for 20 min. Aliquots (50 tzl) were used to measure kinins released with a kinin RIA [271. The buffer system differences were normalized with overlapping pHs.

Inhibition study. The effect of various trypsin inhibitors on the purified rat prostate kallikrein was examined using rat low M r kininogen as the substrate. 50 tzi of enzyme (1 mg/ml) was incubated with 50 ~1 of inhibitors of various concentrations in 0.1 M sodium phosphate (pH 8.5) at 37°C for 30 min, Rat low M~ kininogen (3 #g) in 400 #1 was added to the solution, mixed and incubated at 37°C for 1 h. Released kinins were measured by a kinin RIA [27].

Results

Purification. Rat prostate kallikrein was purified as described in Materials and Methods. Table I summa- rizes the purification scheme of this enzyme.

Molecular mass. Fig. 1 shows the SDS-PAGE profile of the purified prostate kallikrein and other kallikrein gene family members under reducing conditions. Prostate kallikrein migrates as a major band with a molecular mass of 32 kDa and two minor bands of 18 and 14 kDa (lane 1). Under the same conditions, tonin (lane 2) and tissue kallikrein (lane 3) migrate as a single polypeptide chain with molecular masses of 32 and 38 kDa, respectively, while T-kininogenase (lane 4) and esterase A (lane 5) each consist of two bands of 22 and 6 kDa. In the absence of reducing reagent, prostate kaUikrein appears as a single band on SDS-PAGE with a molecular mass of approx. 28 kDa (data not shown). Furthermore the purified prostate kallikrein $3 is not contaminated with other kallikrein gene family enzymes since Western blot analysis showed that the prostate kallikrein $3 was not recognized by specific antibodies to other kallikrein gene family members,

Mrx10 -3

97 m !

66" |

43 m

2 1 ~ , m D , . . , .

1 4 ~ '

1 2 3 4 5 Fig. I. SDS-PAGE of rat prostate kallikrein and related kallikreins. 4 /zg of each enzyme was electrophoresed on a 7.5-15% SDS-polyacrylamide slab gel in a Tris-glycinc buffer system (pH 8.8). Coomassie brilliant blue protein staining shows: rat prostate kallikrein (lane I), tonin (lane 2), rat tissue kallikrein (lane 3), T-kininoGenase (lane 4), esterase A (lane 5). Molecular weight standards: rabbit muscle phosphorylase b, 9741111: bovine serum albumin, 66200; hen egg white ovalbumin, 42699: bovine carbonic anhydrase, 31000;, soybean trypsin inhibitor. 21500; and hen egg white ly~zyme, 144110.

such as tissue kallikrein, T-kininogenase or tonin (data not shown).

NH2-terminal amino acid sequence analysis. Fig. 2 shows the NH2-terminal sequences of the 32 kDa intact protein and both the light and heavy chains of the prostate kallikrein. These sequences are aligned with the translated amino acid sequences encoded by tonin [28], rat tissue kallikrein, P1 [29], RSKG7 or KI [12-13]. The NHz-terminal sequences of the light and heavy chains are identical to the derived protein sequence encoded by $3 prostate kallikrein eDNA [29]. The NH2-terminal sequence of the 32 kDa intact polypeptide and the 14 kDa light chain are identical

TABLE !

Purification of rat prostate kallikrem

One enzyme unit is the amount of the enzyme which releases I pg angiotensin II from angiotensin I at 37°C in 30 rain.

Purification step Total protein Enzyme activity Specific activity - Purification Recovery (rag) (unit) (unit/rag) factor (%)

Prostate extract 435 1.14 0.0026 1 100 DEAE-Sepharose CL-6B 12.2 0.92 0.076 29 80.7 HPLC gel filtration 2.62 0.40 O.151 58 35.0

Pzostate kalllkzein

(~ntact or light chain)

Translated $3 mRNA

Tonin

Tissue kalllkrein

Pl

RSKG? or K1

i 5 I0 15

VVGGYNCETNSQPWQVA

I i I I t I I I I I I I I I I I I VVGGYNCETNSQPWQVA

IVGGYKCEKNSQPWQVA

VVGGYNCEMNSQPWQVA

I I GGFN~EKNSQPWQVA

VI GGYKCEKNSQPWQVA

Prostate kalllkrein

(heavy chain)

Translated $3 gh~lqA

Tonin

Tissue kallikreie

PI

RSKG7 or K1

8 8 9 5 1 0 5

A Y D H N N D L M L L H L S K P A D I T

I I I I I I I I I I I I I I I I I I I I AYDHNNDLMLLHLSKPADIT

VHDHSNDLMLLHLSEPADIT

GDDYSNDLMLLHLSQPADIT

GNDYSNDLMLLHLKTPADIT

GDDHSNDLHLLHLSQPADIT

Fig. 2. N-terminal amino acid ~quencc comparisons of rat prostate kallikrein and kallikrein gene family enzymes. The partial amino acid sequence of the prostate kallikrein (top line) was determined in the present work, The translated amino acid sequences of the kallikrein family members are from [10.14.15,29]. The amino acid residues dre

numbered taking the amino terminus of rat tissue kallikrein as I.

(data not shown). The results indicate that the 14 kDa light chain and ~he 18 kDa heavy chain are the prod- ucts at a single cleavage of the 32 kDa protein between amino acid residues Arg-87 and Ala-88 in the kallikrein autolysis loop [30]. Partial amino acid sequence analysis showed that the prostate kallikrein shares 75-84% identity with other kallikrein-like enzymes.

lsoelectric focusing, lsoelectrofocusing of the purified kallikrein-related enzymes was performed on a 0.5 mm thin-layer polyacrylamide slab gel in a pH gradient of 3.5-10.0. Fig. 3 shows multiple bands for human tissue kallikrein (lane 1), rat tissue kallikrein (lane 2), tonin (lane 3), T-kininogenase (lane 4) and rat prostate kailikrein (lane 5). Prostate kallikrein has two major bands with isoelectric points ranging from 7.30-7.45 (lane 5). Rat prostate kallikrein, similar to human prostate-specific antigen with a p l of 7.41, is a basic protein while tissue kallikrein and other gene family members are acidic proteins.

Enzymatic characterization. Table 11 shows the kinin-releasing and angiotensin ll-releasing activities of rat prostate kallikrein and three kallikrein gene family

313

::,:/(::: :

i

10.0"

9.0"

&O' Q

~ 7 . 0 '

Z &O"

&O"

4.O

0 2 4 6 8 10 12 14 16 18 20

Sl ice Fig. 3. lsoelectric fl)cusing of rat prostate kallikrein on a polyacrylamide slab gel. For detailed procedures, see Materials and Methods. Coomassie brilliant blue protein staining shows: human urinary kallikrcin (lane I), rat salivary tissue kallikrein (lane 2), tonin (lane 3), rat T-kininogena~ (lane 4) and rat proslatc kallikrein (lane 5).

The corresponding pH values are shown at the bonom.

members, tissue kallikrein, tonin and T-kininogcnasc, using natural protein substrates including rat low M r kininogen, T-kininogen, bovine low M, kininogen~ angiotensin ! and angiotensinogen. The rat prostate kallikrein cleaves rat low M, kininogen while it exhibits minimal activity in processing T-kininogen and bovine low M, kininogen. Tissue kallikrein shows high specific activities toward both rat and bovine low M, kininogens, and has 5% of T-kinin-releasing activity as compared to T-kininogcnase. T-kininogenase shows highest activity toward T-kininogen, and it also displays high

TABLE Ii

Kinin- and angiotensin H-releasing actirities o f rat prostate kallikrein and related enzymes

Specific activity is expressed as /zg product formed/mg protein per 30 min. Values represent mean 4-_ S.D. (n = 3 or 4). Concenlration of purified enzymes were calculated from extinction coefficient (E,~).,.i,~; ) [23], which are 1.3 for prostate kallikrein, 1.5 for tissue kallikrein, 1.0 for tonin and 3.5 for T-kininogena~.

Substrate Specific activity

prostate kallikrein tissue kallikrein tonin T-kininogenase

Rat low bl r kininogen 0.20 + 0.03 34.47 + 4.14 0. ! 5 + 0.06 62.43 _ 9.57 T-kininogen 0.02 _+ 0.02 5.54-+ 0.49 0.00 :h 0.00 120.92 _+ 31.89 Bovine low M, kininogen 0.04_+0.02 30.03_4-5.02 0.18=1:0.22 20.37_+ 6.12 Angiotensin I 0.37_+0.05 0.01 _+0.01 9.88_+ !.62 0.61 _+ 0.21 Angiotensinogen 0.50_+0.14 0.00-+ 0.00 8.71 + 1.11 1.03-+ 0.04

a Specific activity is expressed as pg product formed/mg protein per 30 min. Values represent mean + S.D. (n = 3 or 4).

314

t t 3

:~ 100-

i~ 80

< 6o bJ) .=_

• 0 g i • | " ¢ " i

4.5 5.5 6.5 7.5 8. 5 9.5 1 0.5 pH

Fig. 4. Optimal pH determination of rat prostate kallikrein. For detailed procedures, see Materials and Methods. The kinin-releasing activity of rat prostate kallikrein using rat low M, kininogen sub-

strafe was expressed as the percentage of the activity at pH 8.5.

activity toward both rat and bovine low M r kininogens. Contrarily, tonin shows low activity toward rat and bovine low M~ kininogens, and has no activity in hydrolyzing T-kininogen. The prostate kallikrein releases angiotensin I! from both angiotensin I and angiotensinogen substrates. Its specific activity toward angiotensin I and angiotensinogen is about 26- and 15-fold lower than that of tonin. T-kininogenase exhibits angiotensin ll-releasing activity comparable to that of prostate kallikrein $3.

Table 111 shows the comparison of the hydrolysis of tripeptide substrates, D-Pro-Phe-Arg-MCA, o-Pro-Phe- Arg-pNa, D-Vai-Leu-Arg-pNa and o-lle-Pro-Arg-pNa by prostate kallikrein, tissue kallikrein, tonin and T- kininogenase in the initial velocity measurement. The prostate kaUikrein shows very low peptidase activity in hydrozying these kallikrein substrates except for D-lle- Pro-Arg-pNa. Tissue kallikrein has high activity toward all four peptides in the following order: D-Pro-Phe- Arg-pNa > o-Val-Leu-Arg-pNa > D-Pro-Phe-Arg- MCA > o-lle-Pro-Arg-pNa. Tonin exhibits relatively low activity towards these substrates in a order: o-Pro- Phe-Arg-MCA > o-lle-Pro-Arg-pNA > D-VaI-Leu- Arg-pNa > o-Pro-Phe-Arg-pNa. It is surprising to find that T-kininogenase has the highest activity in the

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Rat Prostate Kallikrein (ng/tube)

Fig, 5. Log-logit transformation of a typical radioimmunoassay standard curve of rat pro.~tate kallikrein (e), serial dilutions of rat prostate extract ( • ) and rat submandibular extract (A). The prostate kallikrein standard curve ranged from 4(I pg to 5.0 ng. This parallelism indicates immunological identity between the kallikrein measured in the rat prostate and submandibular gland extract and the purified prostate enzyme. B / B o is the amount of bound radioactiv- ity in the presence, divided by that in the absence, of unlabeled kallikrein using rabbit anti-(rat prostate kallikrein) antiserum, the

ratio being exprcs~d in percentage terms.

hydrolysis of Pro-Phe-Arg-pNa while it displays minimal activity toward Pro-Phe-Arg-MCA with identical P3-P2-Pn-residues.

The kinin-releasing activity of the prostate kallikrein from rat low M r kininogen substrate is pH dependent with very low activity at pH below 7.5 and increasing activity toward alkaline pH with an optimum of 8.5 (Fig. 4). Its kinin-releasing activity is inhibited by leu- pcptin, antipain and benzamidine in a dose-dependent manner. The activity is slightly inhibited by soybean trypsin inhibitor, but not inhibited by a wide range concentrations of lima bean trypsin inhibitor or PMSF (Table IV).

Det'eiopment of prostate kailikrein radioimmunoassay. The antibody titration cu.rve of the prostate kallikrein shows that in the absence of competitive antigen, the antiserum titer, measured by its ability to bind 35% of nzSl-labeled antigen, was 1:20000. By using different combinations of 25% poly(ethylene gly-

TABLE I11

Peptidase actici~, o] rat prostate kallikrein and related enzymes

Initial velocity of enzyme activities was measured at 23°C in 0.05 M Tris-HCI (pH 8.0) as described in Materials and Methods. Specific activity is expressed as mol of substrate hydrolyzed/min per mol enzyme. Values represent mean ± S.D. (n = 3 or 4).

Substra,te Specific activity

prostate kallikrein tonin tissue kallikrein T-kininogenase

Pro-Phe-Arg-MCA 0.25±(I.22 10.3+_0.7 627.3±20.0 73.0± 2.7 Pro-Phe-Arg-pNa 0.75 + 0.34 2.2 ± 0.3 781.7 + 69.6 1372.6 ± 18.7 VaI-Leu-Arg-pNa 0.65 _+ 0.01 6.3 + 0.21 678.4 ± 15,6 401.5 ± 49.4 Ile-Pro-Arg-pNa 5.69± 1.56 9.4+ 1.2 !14.6± 7.8 831.0+ 61.9

TABLE IV

l~'fft, t't.~ o f t~'p.~br hthibitorx on the kil~in-rt'leasing at'lit'it)" o f rat prostate kallikrcin

IC.~t} is the inhibitor concentration giving 51J~ inhibition of prostate kallikrcin's kinin-releasing activity. The details of inhibition study were described under Materials and Methods. Purified rat low M, kininogen (3/zg) was used as the subsirate.

Inhibitor IC~

Leupeptin 1.8" 10-* M Antipain 1.0.10-4 M Benzamidine 9.4.10 -'~ M Soybean trypsin inhibitor 3.8" l0 5 M Lima bean trypsin inhibitor No inhibition PMSF (I raM) No inhibition

col) and 1% bovine y-globulin, it was determined that the optimal combination is 25% poly(ethylene glycol) (0.3 ml) and !% bovine y-globulin (0.1 ml) for separat- ing the antibody-bound enzyme from the free form. This chosen combination of poly(ethylene glycol) and bovine y-globulin yields < 8% nonspecific binding and an intra-as~y error of < 6% and an inter-assay error of < 9%. Fig. 5 shows the parallelism between serial dilutions of rat prostate and submandibular gland extract and the prostate kallikrein standard curve, which ranges from 40 pg to 5.0 ng. The parallellism indicates immunological identity between the enzyme measured in the prostate and submandibular gland and the purified prostate kallikrein standard. In this assay system, there is less than I% cross-reactivity with other kailikrein gene family enzymes including tonin (0.3%), tissue kallikrein (0.3%) and T-kininogenase (0.13%). Prostate extract contains the highest concentration of prostate kallikrein with a specific activity of 23.78 + 2.62 # g / r a g protein (n = 5). Submandibular gland also contains high levels of this enzyme with a specific activity of 12.29 + 2 .25/zg/mg protein (n = 5). Detectable levels of the prostate kallikrein were found in the per- f u n d tissue extracts of pancreas, pituitary and kidney and serial dilutions of these tissue extracts also showed parallelism to the standard prostate enzyme in the RIA (data not shown). However, specific activities of the prostate kallikrein in the pancreas, pituitary and kidney are at least 1000-fold lower than those in the prostate and submandibular gland.

Discussion

The present report describes the purification and characterization of a prostate enzyme which belongs to the rat kallikrein multigene family. NH 2-terminal amino acid sequence of the purified prostate kallikrein matches completely wi:h a eDNA ($3) isolated from a rat salivary eDNA library [29] and indicates that the prostate kallikrein is encoded by $3 mRNA. In con-

315

trast to other kallikrein gene family enzymes including rat tissue kallikrein, tonin and T-kininogenasc, the prostate kallikrein is a basic protein with p l of 7.3-7.45. Using a highly sensitive and specific RIA developed for the prostate kallikrein, the enzyme was found at highest levels in the prostate gland and with a 2-fold lower levels in the submandibular gland. The enzyme can also be detected by the RIA at very low levels in per fu~d tissue extracts from the pancreas, kidney and pituitary. Whether low levels of the prostate kallikrein pre~nt in these important organs are attributed to gone expression has yet to be determined.

Rat prostate kallikrcin shares 75.3 and 84.2% sequence identity with rat tissue kallikrein (PS) and tonin (RSKG 5 or $2), respectively (Fig. 2). Amino acid residues His-57, Asp-102 and Ser-195 of the serine proteinase charge relay system are conserved in the prostate enzyme. The prostate kallikrein encoded by $3 mRNA has the same $2 subsite specificity as tonin with His-99 and Gly-215 [29] and exhibits tonin-like activity by releasing angiotensin II from angiotensinogen and angiotensin ! substrates and hydrolyzing tripeptide substrate D-lle-Pro-Arg-pNa (Table Ill). At the corresponding positions, Chen and Bode [31] have proposed that a principal determinant in the specific cleavage of the kininogen precursor by tissue kallikrein is the presence of a hydrophobic sandwich formed by Tyr-99 and Trp-215 that facilitates the binding of bulky, hydrophobic residues in the substrate adjacent to the cleavage site. The prostate kallikrein, like other kallikreins, has the conserved Asp-189 residue which is characteristic of serine proteinases with a trypsin-like cleavage preferrcncc.

Although the rat prostate kallikrein and other kallikrcin gone family members share cxtcnsivc sequence similarity, these enzymes differ dramatically in substrate specificities (Tables It and Ill). in contrast to other kallikrein-like enzymes, the prostate kallikrcin has very low peptidase activity in hydrolyzing synthetic tripeptide substrates such as D-Pro-Phe-Arg-MCA, O- Pro-Phe-Arg-pNa and o-VaI-Leu-Arg-pNa. While it has the comparable activity with tonin in hydrolyzing D-lle-Pro-Arg-pNa. It is intriguing to find that the specific activity of T-kiniogena~ toward D-Pro-Phe- Arg-pNA is 20-fold higher than that toward D-Pro-Phe- Arg-MCA. The results provide strong evidence that peptide or protein substrate subsite P'I , P'2, P'3 and its extended moieties are important in determining the substrate specificity of kallikrein gene family enzymes as described in previous studies [32].

The prostate kailikrein and other kallikrein gone family enzymes are similar in their basic structures while they differ in substrate and /o r inhibitor binding sites to achieve different functional capabilities at the target tissues. The kinin-relcasing activity of the prostate kallikrein is resistant to inhibition by PMSF

316

(Table IV) and to labeling of active-site serine residue by 14C-DFP (data not shown). Differences of kallikrein gene family enzymes in susceptibility to inhibition by PMSF may represent subtle differences in the confor- mation of these enzymes. Based on X-ray diffraction data, the tertiary structures of porcine pancreatic kallikrein and rat tonin are remarkably similar [33]. The most conserved regions line the substrate binding pocket while the variable regions tend to lie on the exterior of the molecule. The structure and function relationship of prostate kallikrein will be further characterized.

The prostate kallikrein is the major kallikrein gene family enzyme expressed in the rat prostate gland. Previous studies showed that $3 gene expression de- creased after castration and increased with androgen treatment at both mRNA and protein levels [34-35] Using a highly sensitive and specific RIA for the prostate kallikrein, we have also found that castration results in a decrease of immunoactive prostate kallikrein levels in a time-dependent manner and tes- tosterone replacement but not cortisol treatment in the castrated rats restores the enzyme levels (data not shown). The regulation of its expression in prostate gland by androgen could be a primary effect of androgen on prostate kallikrein gene transcription or sec- ondary to cellular regression or cell death post-castration. The specific hormone action could be achieved through a receptor s~tem or through organization of genomic hormone responsive elements. As the hormonal regulation of $3 is prostatic-specific, the physiological function of the prostate kailikrein may be related to androgen levels.

The biological function of the rat prostate kallikrein is not known. It may represent a counterpart of the human prostate-specific antigen (PSA) or hGK-1 gene product [36-38] as the rat and human prostate kallikreins share several characteristics. Firstly, both of them are expressed at high levels in the prostate gland; secondly, they are basic proteins; and thirdly, they have no chymotryptie (tyrosine-cleaving) or elastolytic (alanine-eleaving) activity. Whether rat prostate kallikrein functions to process seminal vesicle protein as does human PSA, remains to be determined.

Acknowledgements

This work was supported by the National Institute of Health grants HL 29397 and DE 0973.

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Biochemical characterization and substrate specificity of rat prostate kallikrein (S3): comparison...

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