ISOLATION AND CHARACTERIZATION OF FOUR PEPTIDE HYDROLASES FROM THE CYTOSOL OF RAT INTESTINAL MUCOSA

Transcription

1 GASTROENTEROLOGY 72:93-100, 1977 Cpyright 1977 by The Williams & Wilkins C. Vl. 72, N.1 Printed in U.S.A. ISOLATION AND CHARACTERIZATION OF FOUR PEPTIDE HYDROLASES FROM THE CYTOSOL OF RAT INTESTINAL MUCOSA CAROL M. SCHILLER, PH.D., TAl-IN HUANG, AND WILLIAM D. HEIZER, M.D. Department f Medicine, University f Nrth Carlina, Chapel Hill, Nrth Carlina The high speed supernatant fluid prepared frm rat intestinal mucsa was subjected t in-exchange chrmatgraphy n diethlaminethyl-cellulse eluted with a linear gradient f sdium chlride (0 t 0.27 M). Assay f eluted fractins fr Phe-Gly hydrlase activity revealed fur distinct peaks f enzyme activity. These cytsl enyzmes have been designated I, II, III, and IV in rder f their elutin frm the clumn. Examinatin f the substrate specificity f the fur enzymes by use f 20 mm peptide cncentratins indicated the mst discriminating substrates fr the fur enzymes were Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide, respectively. The mean distributin f the recvered peptide hydrlase activities against these substrates amng the fur enzymes I, II, III, and IV was 96.1, 1.4, 1.7, and 0.8%, respectively, fr Leu-Gly-Gly; 0.6, 96.4, 2.4, and 0.6% fr His-Met; 0, 0, 95.8, and 4.2% fr Ser-Phe; and 20.8, 19.8, 5.6, and 53.8% fr leucine amide. In-exchange chrmatgraphy resulted in increases in specific activity f 19-, 19-, 46-, and 3.5-fld fr enzymes I, II, III, and IV, respectively. The activity f all fur enzymes, but especially III and IV, were stabilized by the presence f 150 /-LM dithierythritl. Activity f each f the fur enzymes was decreased 79 t 100% by 1 mm ethylenediaminetetraacetate, HgCl 2, 1,10-phenanthrline, r 0.5 mm p-hydrxymercuribenzate, except that the activity f enzyme I was decreased nly 15% by ethylenediaminetetraacetate. N significant activatin f the partially purified enzymes ccurred in the presence f 500 /-LM Zn++, C++, r Mg++. The fur enzymes exhibited distinct ph prfiles with ptima at 7.5, 7.5, 8.5, and 8.0 fr enzymes I, II, III, and IV, respectively. Mlecular weights f the fur enzymes determined by gel filtratin n Sephadex G-200 were 58,500, 74,000, 97,500, and 113,000, respectively. All fur enzymes lst mre than 85% f their activity after 1 hr at temperatures f 50 C r higher in sdium phsphate buffer, ph 7.0. The Km values determined with the mst specific substrates fr each enzyme were 0.76,0.44,3.82, and 8.3 mm fr enzymes I, II, III, and IV, respectively. Recent evidence suggests that a significant amunt f sme small peptides are absrbed intact and hydrlyzed by cytsl peptide hydrlases. Adequate understanding f the functin and cntrl f these intracellular enzymes requires knwledge f the characteristics and substrate specificity f individual enzymes. The study described here demnstrates the presence f at least fur cytsl peptide hydrlases with distinct substrate specificities. Substrates almst exclusively hydrlyzed by each f three f the enzymes, and therefre suitable fr assay f each f these enzymes in the presence f the thers, have been identified. Many small peptides are frmed in the intestinal lumen by the actin f prteases and are hydrlyzed t free amin acids during the prcess f absrptin. 2-4 Thus, peptide-hydrlyzing enzymes f the intestinal Received April 5, Accepted July 19, A preliminary reprt f this wrk has been published. 1 Address requests fr reprints t: William D. Heizer, Divisin f Gastrenterlgy, Department f Medicine, University f Nrth Carlina, Chapel Hill, Nrth Carlina This wrk was supprted by Research Grant R01 AM frm the United States Public Health Service. Dr. Schiller's present address is Natinal Institute f Envirnmental Health Sciences, Research Triangle Park, Nrth Carlina mucsa play an essential rle in prtein digestin analgus t the rle f disaccharidases in carbhydrate digestin. Unlike disaccharidases, which are virtually limited t the brush brder, peptide hydrlase activities are present in the cell cytsl as well as in the brush brder. 5-8 It has been shwn that cytsl and brush brder peptide hydrlase activities are attributable t different enzymes. 9, 10 Althugh the relative imprtance f the brush brder and cytsl enzymes fr digestin f peptides derived frm intraluminal prteins is still unclear, current evidence indicates that cytsl enzymes may play an imprtant rle, especially in the digestin f dipeptides. 1l - 16 Investigatin f the develpment, cntrl, and func- 93

2 94 SCHILLER ET AL. Vl. 72, N.1 tins f cytsl peptide hydrlases has been hampered by inability t measure the activity f individual enzymes in crude cytsl preparatins. Activities can and have been measured by means f substrates chsen at randm frm the mre than 500 dipeptides and thusands f tripeptides cmprising the cmmnly ccurring amin acids. Hwever, results f such measurements are meaningless fr many purpses because the number f enzymes hydrlyzing each substrate is unknwn. Sme prgress in islating and characterizing the intestinal cytsl enzymes has been made. Single peptide hydrlases have been islated frm the mucsa f several animals,17'20 and studies, including starch gel electrphresis, have shwn that cytsl preparatins frm rat and guinea pig intestinal mucsa each cntain several peptide-hydrlyzing enzymes. 10, Hwever, the number f sluble peptide hydrlases present in the intestinal mucsa and their substrate specificities have nt been reprted fr any species, The purpse f the study reprted here was t separate the cytsl peptide hydrlases f rat mucsa and t investigate their substrate specificities in rder t identify, if pssible, substrates that are hydrlyzed exclusively by each enzyme. Experimental Prcedure Tissue preparatin, Sprague-Dawley rats (200 t 250 g males) were fasted vernight, killed by a blw n the head, and the mucsa frm the entire small intestine was harvested by scraping. The cytsl fractin was prepared by hmgenizing the mucsal scrapings in ice-cld 5 mm sdium phsphate buffer, ph 7.0, (2 ml per g f wet weight) in a Ptter-Elvehjem hmgenizer and centrifuging fr 1.5 hr at 86,000 x g at 4 C, Enzyme assays. The extent f hydrlysis f the varius substrates was determined by use f a clrimetric assay fr liberated L-phenylalanine, L-methinine, r L-Ieucine as previusly described,25 One unit f hydrlase activity is defined as that amunt which, under standard cnditins, catalyzes liberatin f 1 /-Lmle f L-amin acid in 1 min. All assays were perfrmed in duplicate. Results f duplicate determinatins did nt differ by mre than 10% and values recrded are means f tw results. The ph ptima f the enzymes were determined using a series f 0.5 M Tris maleate and Tris-HCI buffers. All subsequent experiments were perfrmed at the ph ptimum determined fr each enzyme. Fr studies f ph ptima and fr thse invlving additin f metal ins t the reactin mixture the incubatin reactin was stpped by immersin in biling water fr 3 min t avid pssible errr resulting frm incmplete inactivatin f peptide hydrlases by 1,10-phenanthrline under these nnstandard assay cnditins. The high vltage paper electrphresis methd used t screen enzyme preparatins fr activity against large numbers f peptide substrates was as previusly described by Heizer and Laster26 with sme mdificatins, The incubatin medium cntained 0,7 /-Lmle f Tris-HCI buffer, ph 8.0, 0.5 /-Lmle f substrate, and enzyme in a ttal vlume f ml. Incubatin time was 30 min fr dipeptides and 90 min fr plypeptides. Sensitivity f this semiquantitative assay is nt the same fr every substrate. In mst cases release f as little as 0.2 t 0.5 nmle f free amin acid during the incubatin wuld have been recrded as trace hydrlysis. L-Leucyl-i3-naphthylamidase activity was determined by the methd f Martinik et al. 27 Clumn chrmatgraphy. Standard type 70 diethylaminethyl (DEAE)-cellulse (apprximately 0.90 meq per g) equilibrated with 5 mm sdium phsphate buffer, ph 7.0, cntaining 150 /-LM dithierythritl (DTE) was prepared and packed by air pressure in 2.5- by 30-cm clumns as described by Petersn. 28 Clumns were eluted at 4 C by upward flw maintained with a peristaltic pump at 70 ml per hr. Fractins were cllected atumaticallyat 18-min intervals. Fresh clumns were prepared fr each new sample and charged with 5 t 6 ml f supernatant fluid btained frm the mucsa f 1 rat. Sephadex G-200, 40- t 120-/-L particle size, was hydrated in 0.2 M sdium phsphate buffer, ph 7.0, fr 3 days and then packed at lo-cm water pressure in a 1.5- by 60-cm clumn. Applicatin f 0.36 ml f sample was fllwed by 0.36 ml f 10% sucrse. Elutin was at 4 C by upward flw maintained with a peristaltic pump at 5.5 ml per hr. Fractins were cllected autmatically at 17-min intervals in tared tubes, and vlumes f each fractin were calculated frm the net weight and measured specific gravity f the elutin buffer. Prtein cncentratin in the fractins was measured by absrbance at 280 nm and enzymes were lcalized by assay. The clumn was calibrated with 0.36-ml samples f Blue Dextran 2000 (10 mg per ml), valbumin and ribnuclease A (6 mg f each per mi), and aldlase and chymtrypsin (6 mg f each per ml). A plt f the Kav versus the lg f the mlecular weights fr the prtein standards yielded a straight line frm which mlecular weights f the islated peptide hydrlases were estimated. Miscellaneus. Prtein cncentratin was measured by the methd f Lwry et al. 29 with crystalline bvine albumin as the standard. The prtein elutin prfile frm the DEAE-cellulse clumns was mnitred at 280 nm. Prteins in clumn fractins were cncentrated n Amicn ultrafiltratin cells fitted with UM-I0 membranes. Materials. Peptides were btained frm Bisynthetika (Oberdrf, Switzerland), Bachem Cmpany (Marina Del Rey, Calif.), and Sigma Chemical Cmpany (St. Luis, M.). Lyphylized Crtalus adamanteus venm and -dianisidine dihydrchlride were btained frm Sigma Chemical Cmpany, free amin acids frm Mann Research Labratries (New Yrk, N. Y.), 1,1O-phenanthrline frm Fisher Scientific Cmpany (Pittsburgh, Pa.), DTE frm Cycl Chemical Crpratin (Ls Angeles, Calif.), and hrseradish perxidase frm Wrthingtn Bichemical Crpratin (Freehld, N. J.). All ther chemicals used were f reagent grade. Amin acids and peptides used shwed n, r in a few cases, minimal and insignificant, cntaminatin by ther ninhydrin-reactive material as determined by the high vltage paper electrphresis methd. 26 All free and peptide-bund amin acids were L-cnfiguratin. Results Stabilizatin f cytsl peptide hydrlases. Instability f cytsl peptide hydrlases in slutin is well knwn, and in the present study Gly-Phe hydrlase activity in a supernatant preparatin decreased mre than 90% in 48 hr at 4 e. When the supernatant cntained 15 JLM DTE, Gly-Phe hydrlase was nt immediately inactivated and activity decreased nly 15% in 48 hr. Prgressively higher cncentratins f fresh DTE caused a prgressive fall in Gly-Phe hydrlase activity in fresh supernatant slutin (85% decrease in the presence f 1 mm DTE), nt all f which culd be accunted fr by the deleterius effect that DTE als had n the clrimetric assay (38% decrease in absrbance prduced by standard amunt f free phenylalanine at 1 mm DTE). Inhibitin f cytplasmic Gly-Phe hydrlase by DTE is reversible as indicated by the fact that enzymes stred at 4 e in the presence f

3 January 1977 CYTOSOL PEPTIDE HYDROLASES t 250,llM DTE shwed a gradual rise in Gly-Phe hydrlase activity with time, apparently due t the slw spntaneus xidatin f the DTE. This increase was fllwed by a decrease in activity due t inactivatin f the enzyme in the absence f sufficient reduced DTE. Supernatant fluid cntaining less than 30,llM DTE shwed nly a decline in Gly-Phe hydrlase activity with time. On the basis f these results, and the fact that many experiments required lnger than 48 hr fr cmpletin, buffers used fr enzyme preparatin and separatins were made 150,llM with DTE. Cytsl Gly-Phe hydrlase activity was nt stabilized by the presence f 10,llM ethylenediaminetetraacetate (EDTA), ZnC1 2, r cysteine. Islatin f cytsl peptide hydrlases. Chrmatgraphy f the high speed supernatant fluid n DEAE-cellulse by elutin, first with 5 mm phsphate buffer and then with a linear sdium chlride gradient, gave fur well separated peaks when the effluent fractins were assayed fr Phe-Gly hydrlase activity (fig. 1). The fur peaks are designated enzymes I, II, III, and IV, based n the rder in which they elute frm the clumn. The mst active fractins f each f the fur peaks were pled and equal vlumes f the pls were tested fr their ability t hydrlyze 85 peptide substrates by use f a semiquantitative, high vltage, paper electrphresis assay. The results (table 1) indicate that certain peptides are hydrlyzed almst exclusively by each enzyme such as Gly-Gly-Gly, Leu-Gly-Gly, and Phe-Gly-Gly by enzyme I; His-Met, His-Gly, Lys-Glu, and Lys-Lys by enzyme II; Gly-Phe, Gly-Pr, Leu-Val, Leu-Ser, Ser-Phe, and thers by enzyme III; and leucine amide by enzyme IV. Quantitative assays f DEAE-clumn fractins with Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide cnfirmed the usefulness f these substrates fr identifying individal enzymes (fig. 2). Only leucine amide was significantly hydrlyzed by mre than ne enzyme. L-Leucyl-!3-naphthylamidase hydrlysis gave a pattern similar t that f leucine amide in that the greatest activity was in peak 4, but peaks 1 and 2 als shwed significant activity. High speed supernatant prepared by hmgenizing mucsa in 0.25 M sucrse buffered with 5 mm phsphate shwed a pattern n DEAE-cellulse chrmatgraphy that was essentially identical t that btained when supernatant was prepared with 5 mm phsphate buffer alne. Recvery and purificatin. In fur separate experiments, supernatant prepared frm the intestinal mucsa f individual rats was subjected t DEAE-cellulse chrmatgraphy. The supernatant and the clumn fractins were assayed quantitatively fr hydrlysis f each f the fur mst discriminating substrates. The percentage f ttal recvered activity against individual substrates that is attributable t each f the fur enzymes is shwn in table 2. The data demnstrate the high degree f specificity f each enzyme fr ne f the fur substrates and als shw the upper limits f pssible cntaminatin f each peak by the ther three. Fr example, data fr Leu-Gly-Gly hydrlysis shw that mean cntaminatin f enzyme II, III, and IV preparatins by enzyme I des nt exceed 1.4, 1.7, and 0.8%, respectively. Based n assays f whle supernatants, ttal percentage f recveries (±1 sn) f the hydrlase activities against Leu-Gly-Gly, His-Met, Ser-Phe, leucine amide, and Phe-Gly were (±2.6), 64.1 (±17.5), E c::: 00.6 f() 10 z «- 0.5 ENZYMES ISOLATED FROM SUPERNATE OF RAT INTESTINAL MOCOSA BY DEAE-CELLULOSE CHROMATOGRAPHY PHE-GLY HYDROLASE ACTIVITY AND PROTEIN CONCENTRATION In 0 3 >-!::: 02 w 0.1 (/) «...J 0:: :I: I, " II " II " I I I I t :, I! + /' I \ ' : I,f( j..--t' f /' "i ' ,/',/' /',/' /' I I I I I I! 1 I I I I I 5' FRACTION NUMBER 02 :::: " CT w E (J c 0.1 z., 06 E c::: 05 - w 0.4 «03 (/) z iii I- 0 0:: a. FIG. 1. Diethylaminethyl (DEAE)-cellulse chrmatgraphy f mucsal supernatant. Intestinal mucsa (6.0 g frm 1 rat) was hmgenized in 12 ml f 5 mm sdium phsphate buffer, ph 7.0, which was als 150 /LM with respect t dithierythritl (DTE). Five milliliters f the high speed supernatant was applied t a DEAE-cellulse clumn equilibrated with the same buffer mixture. Chrmatgraphy was carried ut as described in Methds. A linear NaCI gradient (- -) was frmed by using tw flasks f similar size and shape, the first cntaining 700 ml f equilibrating mixture and the secnd cntaining 700 ml f this mixture made 0.27 M with NaCI. Prtein cncentratin (e- - -e) f each fractin was estimated by absrbance at 280 nm and Phe-Gly hydrlase activity ( O was determined ) quantitatively. Fractins marked (') were cllected befre initiatin f the NaCI gradient.

4 96 SCHILLER ET AL. Vl. 72, N.1 TABLE 1. Semiquantitative peptide hydrlase assays f cytsl enzymes islated by diethylaminethyl (DEAE)-cellulse chrmatgraphy" Peptide Ala-Phe Ala-Leu Ala-Met Ala-Tyr Arg-Asp Arg-Glu Arg-Lys Arg-Phe Gly-Phe Gly-Gly Gly-Leu Gly-Met Gly-Thr Gly-His Gly-Pr Gly-Lys Gly-Tyr Gly-Val Gly-Ser Gly-Glu Gly-Ile GIn-Leu Gln-Gly a-glu-tyr a-glu-gly a-glu-glu His-Phe His-His His-Leu His-Gly His-Met His-Asp His-Glu Ile-Val Ile-Gly Leu-Phe Leucine amide Leu-Ala Leu-Gly Leu-His Leu-Val Leu-Ser Lys-Leu Enzyme II III IV Peptide % hydrlysis Tr Tr Lys-Glu Lys-Lys Tr Lys-Phe Met-Phe Met-Ser 5 Tr Tr Met-Leu 5 0 Tr Met-Gly 5 0 Tr Met-Ala 0 25 Tr Met-Val Met-Ile Phe-Ile Phe-Phe Phe-Leu Tr 5 Tr Phe-Pr Phe-Arg 25 5 Tr Phe-Val Phe-Ser 5 75 Tr Phe-Gly Tr 5 0 Phe-Ala Pr-Phe Pr-Met Pr-Gly Pr-Leu Pr-Glu Pr-GIn Ser-Met 25 5 Tr Ser-Phe 25 Tr Tr Thr-Phe 025 TrTr Thr-Leu 25 Tr 0 Thr-Met 25 Tr 0 Tyr-Gly Val-Met 5 OVal-Leu 25 Val-Phe 0 25 Tr Tr Tr Tr 25 Tr Gly-Gly-Gly Gly-Gly-Phe Phe-Gly-Gly Leu-Gly-Gly Lys-Gly-Gly Gly-Gly-Gly-Gly Enzyme II III IV % hydrlysis Tr 5 Tr 0 0 Tr Tr Tr Tr Tr 50 Tr Tr 25 Tr Tr 0 Tr Tr 5 Tr Tr Tr 25 Tr Tr 5 Tr Tr 5 25 Tr Tr Tr 50 5 Tr Tr 0 Tr 5 Tr 0 Tr Phe-Gly-Phe-Gly 0 Pr-Phe-Gly-Lys 550Tr Tr 0 0 Tr Tr Tr Tr Tr Tr Tr 0 0 a Figures are estimated percentage f hydrlysis recrded as 0 (undetectable), Tr (less than 1 %),5 (1 t 11 %),25 (12 t 37%),50 (38 t 62%), 75 (63 t 86%), and 100 (87 t 100%) (±125.3), 35.6 (±31.6), and 89.3 (±18.0), respectively. The riginal supernatants, stred at 4 C, were assayed at the same time as the clumn fractins, apprximately 24 hr after each rat was killed. The large percentage f recvery f Ser-Phe hydrlase activity is in part explained by a mre rapid lss f enzyme III activity in the whle supernatant than during chrmatgraphy, even thugh 150,lIM DTE was present initially in bth circumstances. This methd f determining recvery, 6 FRACTION NUMBER FIG. 2. Substrate specificity f cytsl peptide hydrlases separated n diethylaminethyl (DEAE)-cellulse. Cytsl prteins were chrmatgraphed as described in the legend fr figure 1 and fractins were assayed quantitatively fr hydrlysis f Leu-Gly-Gly (0--0), His-Met (e ), Ser-Phe ( D and ) leucine, amide (_--_). Absrbance values pltted are nrmalized values calculated by dividing the bserved absrbance by 4.9 fr Leu-Gly-Gly, 0.8 fr His-Met, 6.4 fr Ser-Phe, and 0.04 fr leucine amide. Fractins marked (') were cllected befre initiatin f the NaCI gradient. The small peak at fractin 5' is at the breakthrugh vlume fr the clumn and prbably represents unbund prtins f ne r mre f the islated enzymes. TABLE 2. Recvery f peptide hydrlase activities in the fur cytsl enzymes islated by diethylaminethyl (DEAE)-cellulse chrmatgraphya Hydrlase activity Enzyme Leu-Gly-Gly His-Met Ser-Phe Leucine amide II III IV 96.1 (6.2) 1.4 (1.6) 1.7 (3.1) 0.8 (1.5) a Mean percentages (1 SD). 0.6 (0.7) 96.4 (0.6) 2.4 (1.6) 0.6 (0.6) % 95.8 (4.9) 4.2 (4.5) 20.8 (6.0) 19.8 (17.6) 5.6 (8.8) 53.8 (17.6) althugh nt ideal, was chsen because it seemed best t assay bth the crude supernatant and the islated enzymes in the presence f DTE, and the 150,uM DTE initially present nt nly inhibits the peptide hydrlase activity, but als interferes smewhat with the quantitative assay. Hwever, within apprximately 24 hr, sufficient xidatin f the DTE has ccurred t permit nearly ptimal recvery f peptide hydrlase activity as well as an accurate assay. Preliminary experiments indicated that smaller cncentratins f DTEdid nt prtect the peptide hydrlase activities as well ver the perid f time required fr chrmatgraphy and assay. Chrmatgraphy n DEAEccellulse increased the specific activities f enzymes I, II, III, and IV, 19-, 19-, 46-, and 3.5-fld, respectively. The figure ft enzyme IV is lwer than the actual purificatin because leucine amide, the mst discriminating substrate detected fr that enzyme, is als hydrlyzed t a significant extent by enzymes I and II. Cells ther than entercytes cntain sluble peptide hydrlases 10 and the mucsa scraped frm the intestinal surface includes a number f cells ther than

5 January 1977 CYTOSOL PEPTIDE HYDROLASES 97 entercytes. In a single experiment, entercytes islated by the methd f Stern and Jensen 30 were hmgenized and the supernatant was chrmatgraphed. This preparatin yielded fur peaks f peptide hydrlase activity cmparable t thse btained with supernatants f whle mucsa. The percentages f recvered activities, 95.6, 95.0, 94.2, and 83.5% fr Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide in peaks 1, 2, 3, and 4, respectively, were within the ranges determined fr whle mucsal preparatins. ph ptima. Each f the enzymes exhibited a distinct ph prfile when examined fr activity against its mst discriminating substrate ver a ph range f 6.0 t 9.5. Enzyme I had a brad range f activity centered at ph 7.5; enzyme II had a ph ptimum at 7.5 with a sharp decline at ph values greater than 8.0; enzyme III was mst active at ph 8.5 with little decrease even at 9.5; and enzyme IV had a brad range f activity centered at ph 8.0. Mlecular weights. Mlecular weights determined by gel filtratin chrmatgraphy n Sephadex G-200 were 58,500 fr enzyme I, 74,000 fr enzyme II, 97,500 fr enzyme III, and 113,000 fr enzyme IV. Fr these determinatins the enzyme samples and eluting buffer cntained 150 DTE. M Inhibitin and inactivatin. The fur peptide hydrlases were preincubated fr 30 min at 37 C with 5 mm sdium phsphate buffer, ph 7.0, and als with mixtures f the same buffer cntaining 1 mm EDTA, HgCI 2, 1,10- phenanthrline,r 0.5 mm p-hydrxymercuribenzate. As shwn in table 3, the activity f all fur enzymes, which were assayed using the mst discriminating substrates, was markedly decreased by the inhibitrs except that enzyme I was nly slightly decreased by 1 mm EDTA. The presence f 50 and 500 Zn++, M C+ +, and Mg++ in the incubatin media in the absence f metal chelatrs did nt yield significant activatin f any f the enzymes. When the fur enzymes were kept at 0, 20, r 37 C fr 1 hr, 78 t 100% f each activity was recvered. When maintained at 50 and 70 C, less than 15 % f each activity was present after 1 hr. Fr these studies the enzymes were in 5 mm sdium phsphate buffer, ph 7.0, made 150 with M DTE. Enzyme kinetics. Three fractins frm each f the fur peaks, cmprising 60% r mre f the ttal peak activity, were pled and cncentrated. Initial reactin rates were TABLE 3. Inhibitin f cytsl peptide hydrlases Inhibitr" Enzyme II III IV % inhibitin EDTA,lmM HgCl"lmM ,10-phenanthrline, 1 mm phmb,0.5mm aedta, ethylenediaminetetraacetate; phmb, p-hydrxymercuribenzate. determined at final substrate cncentratins between 0.5 and 20 mm, using as substrates Leu-Gly-Gly fr enzyme I, His-Met fr enzyme II, Ser-Phe fr enzyme III, and leucine amide fr enzyme IV. Lineweaver-Burk plts (fig. 3), as well as plts f velcity versus velcity/substrate cncentratin gave straight lines fr each enzyme preparatin. Values f Km (±1 SE), determined by the methd f Hansn et al. 31 using the A. F. Frtune cmputer at the University f Nrth Carlina are: (±0.074) mm fr enzyme I, (±0.113) mm fr enzyme II, 3.82 (±0.365) mm fr enzyme III, and 8.34 (±0.97) mm fr enzyme IV. These values must be cnsidered as prvisinal until it is determined whether each peak cntains nly ne enzyme that hydrlyzes the particular substrate chsen fr assay f that peak. The maximal velcity f hydrlysis (± lsn) f the fur substrates by high speed supernatants f mucsal hmgenates, in units per gram f wet weight f mucsa, is 69.3 (±31.0) fr Leu-Gly-Gly, 24.4 (±O.9) fr His-Met, 34.6 (±34.7) fr Ser-Phe, and 4.1 (±1.7) fr leucine amide. Discussin The rat has been used fr a number f studies f peptide absrptin but there are few reprted attempts t islate and characterize the intestinal cytsl peptide hydr lases frm this animal. The presence f mre than ne peptide hydrlase in the cytsl has been amply dcumented. Rbinsn and Shaw 24 shwed that the cytsl activities respnsible fr hydrlysis f Gly-Ala, Gly-Leu, and Leu-Gly differed in stability. Rbinsn and Shaw 24 and Rbinsn 23 determined that the cytsl f rat intestinal mucsa hydrlyzed Gly-Gly at tw ph ptima, and that the activity at the tw ptima differed in stability. Kim et al., 16 using heat stability and cmpetitin studies, demnstrated that Ala-Gly and Ala-Gly-Gly are hydrlyzed by different cytsl enzymes. Differences in ph ptima and activatin by metal ins have been shwn fr hydrlysis f varius peptide substrates but such differences are nt necessarily evidence f multiple enzymes. Kim, et al. 10 electrph' resed rat intestinal cytsl n starch gel and btained zymgrams that suggest the presence f as many as nine peptide hydrlases, but the enzymes were nt further islated r characterized. Based n the data available it is nt pssible t identify precisely which f the fur enzyme activities islated in this study are respnsible fr hydrlysis f the varius substrates used in the previus studies. The semiquantitative assays in this study did nt shw any marked differences in the pattern f hydrlysis f Gly-Leu and Leu-Gly by the fur enzymes, as bth were primarily hydrlyzed by enzyme III. Hwever, enzyme IV was much mre active against Gly-Leu than against Leu-Gly, and this culd accunt fr the differences bserved by Rbinsn and Shaw.24 The cnclusins f Kim et al. 16 that Ala-Gly and Ala-Gly-Gly are hydrlyzed by different enzymes are als supprted by these semiquantitative assays, which shw that enzyme peak TIl is mst active against dipeptides in which alanine is

6 98 SCHILLER ET AL. Vl. 72, N.1 I II,-,- z :E Z IE 0.3 tl. :E 0.6 IE tl. : E 0.2 c > I/[ LEU-GLY-GLY] (mm") I/[HIS-MET] (mm-') ill, Z 0.6 :E II:.. '" "0 " tl. 0.4 E.5 > 0.2 :::: I,-- :::;; II: '" tl > :::: I/[SER-PHE] (mm-') I/[ LEUCINE AMIDE] (mm-') FIG. 3. Reactin kinetics fr the fur islated peptide hyrlases. Enzymes were islated frm mucsal supernatant n diethylaminethyl (DEAE)-cellulse. Fractins cmprising each enzyme were pled and cncentrated. Initial reactin rates were determined at final substrate cncentratins between 0.5 and 20 mm. Substrates used were Leu-Gly-Gly fr enzyme I, His-Met fr enzyme II, Ser-Phe fr enzyme III, and leucine amide fr enzyme IV. Linear regressin cefficients and 95% cnfidence limits were determined by the methd f Hansn et ai, 31 by use f the A. F. Frtune Bimedical Cmputatin Center cmputer at The University f Nrth Carlina. in the amin-terminal psitin, and enzyme peak I is mst active against tripeptides. The semiquantitative assay used in this study was an invaluable screening device that permitted selectin f a mre limited numer f apprpriate substrates fr quantitative assay. In this way substrates were detected that were highly discriminatry fr three f the fur enzyme peaks islated. Hwever, results f the semiquantitative assay given in table 1 shuld be viewed as nly apprximate. Even using this rapid methd sme lss f activity f these labile enzymes ccurred befre all fur enzymes culd be assayed fr hydrlysis f all the substrates used. Furthermre, the sensitivity f the assay differs fr each substrate, and n attempt was made t determine ptimal cnditins f hydrlysis f each substrate. Als, semiquantitative assays were nt perfrmed n the crude supernatant. Therefre, it shuld nt be cncluded that the cytsl has n hydrlase activity fr substrates such as Gly-Gly, recrded in table 1 as shwing undetectable activity. Many investigatrs , 32 as well as this labratry have demnstrated Gly-Gly hydrlase activity in the cytsl f rat intestinal mucsa. Failure f the authrs t demnstrate activity against tw tetrapeptides is cnsistent with the reprt f Kim et ai.,16 indicating the absence f cytsl activity against substrates lnger than tripeptides. Whether the trace activity against tetraglycine by enzyme I that was bserved is significant must await quantitative studies. The cytsl f rat intestinal mucsa may cntain mre peptide hydrlases than the fur described here. Further purificatin will be required t shw whether each f the fur peaks islated in this study cntain nly ne, r mre than ne, enzyme. On several ccasins chrmatgraphs shwed slight asymmetry in peak 2 (see figs. 1 and 2), suggesting the pssible presence f mre than ne enzyme. In additin, small peaks were smetimes bserved between peaks 1 and 2, but varius substrates used did nt increase the prminence f these peaks mre than that shwn in figure 2. Studies t date indicate rather marked differences in the prperties f intestinal peptide hydrlases between rat and guinea pig. 15 The enzyme islated frm the cytplasm f guinea pig intestinal mucsa by Dnln and Fttrell 21 displays a pattern f substrate specificity that is nt similar t any f thse islated frm rat in the current study. Als, n rat enzyme clsely cmparable t the prlidase activity islated frm the cytplasm f pig intestinal mucsa by Sjstrm et ai. 18 was fund. Althugh nt identical in their substrate specificities, the enzyme islated frm pig intestine by Nren et ai., 19 and that islated frm mnkey small intestine by Das and Radhakrishan,17 bth have brad specificity and tend t resemble enzyme III islated frm rat in the present study. Hwever, the authrs believe that it is premature t cnclude that this r any single intestinal peptide hydrlase is mre imprtant than any ther cytsl r brush brder peptide hydrlase fr digestin f peptides derived frm intraluminal prteins.

7 JaTUUJry 1977 CYTOSOL PEPTIDE HYDROLASES 99 Cathepsins als catalyze the hydrlysis f peptide bnds and are present in many, perhaps all, tissues including the intestinal mucsa There are n detailed studies f the number and substrate specificities f cathepsins in the intestinal mucsa, but it is clear that the peptide hydrlase activities described in this paper are nt attributable t knwn cathepsins, as the cathepsins are lyssmal enzymes which have acid ph ptima and substrate specificities different frm thse f the enzymes described here Based n current evidence, it is generally cncluded that ne physilgical rle f cathepsins and cytsl peptide hydrlases is t catalyze intracellular degradatin f prteins and peptides, including albumin, hemglbin, immunglbulins, enzymes, cllagen fragments, peptide hrmnes, and thers In recent years a number f experiments have suggested that the peptide hydrlases inside intestinal epithelial cells may serve a separate r additinal functin, namely, hydrlysis f small peptides transprted intact acrss the brush brder membrane. At present, there is n evidence t supprt the suggestin (see discussin by Parsns in Reference 47) that different sets f intracellular peptide hydrlases serve these tw functins. The extent f intact peptide transprt under physilgical cnditins is unknwn and is the subject f sme debate Taken tgether, the data are cnvincing that t at least sme extent sme peptides crss the intestinal brush brder intact and are hydrlyzed by cytsl peptide hydrlases. Studies f the rle f intestinal peptide hydrlases have been hampered by lack f knwledge f the substrate specificity f the multiple enzymes present in the cytsl f the mucsa. The current study demnstrates the presence f at least fur mucsal cytsl peptide hydrlases with distinct substrate specificities. Furthermre, substrates highly discriminating fr three f the enzyme peaks, and therefre suitable fr assay f each f these enzymes in the presence f the thers, have been identified. REFERENCES 1. 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