Identification and Characterization of Brush-Border Membrane-Bound Neutral Metalloendopeptidases From Rat Small Intestine

GASTROENTEROLOGY 1986;91:1234-42

Identification and Characterization of Brush-Border Membrane-Bound Neutral Metalloendopeptidases From Rat Small Intestine IN-SUNG SONG, MASAHIRO YOSHIOKA, ROGER H. ERICKSON, SOICHIRO MIURA, DIFU GUAN, and YOUNG S. KIM Gastrointestinal Research Laboratory, Veterans Administration Medical Center, San Francisco, and the University of California, Department of Medicine, San Francisco, California

Neutral metalloendopeptidase enzymes were identified and partially characterized in the brushborder membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine4-methoxy-{3-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thiry-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Received January 19, 1984. Accepted May 5, 1986. Address requests for reprints to: Young S. Kim, M.D., Gastrointestinal Laboratory (151M2), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. This work was supported by grant AM-17938 from the National Institutes of Health and by the Veterans Administration Medical Research Service. I-S. Song is the recipient of an award from the Seoul National University Hospital Fund. S. Miura and M. Yoshioka are recipients of a Fukuzawa Memorial grant from Keio University, Tokyo. Y. S. Kim is a Medical Investigator of the Veterans Administration. A preliminary report of this work was presented at the American Gastroenterological Association in Washington, D.C., May 1983. The authors thank Trish Harrington for expert assistance in the preparation of this manuscript. © 1986 by the American Gastroenterological Association 0016-5085/86/$3.50

Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-{3-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brushborder membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function. Dietary protein is hydrolyzed in the gut by the combined action of pancreatic proteases and peptidases of the intestinal enterocyte. In the brushborder membranes of mammalian small intestinal cells a number of peptidases have been identified and several have been purified. Among these are aminopeptidase N (E.c. 3.4.11.2) (1-7), aminopeptidase A (E.C. 3.4.11.7) (8,9), dipeptidyl aminopeptidase IV (E.C. 3.4.14-) (10,11)' l'-glutamyltranspeptidase (E.C. 2.3.2.2) (12), folate conjugase (13), a carboxypeptidase (10), and peptidyl dipeptidase (E.C. 3.4.15.1) (14). Numerous studies have shown that these enzymes play an important role in the digestion and absorption of protein. Previous studies in our laboratory have demonstrated that significant amounts of 14C-Iabeled rat liver protein were absorbed from rat intestinal Thiry-Vella loops suggesting the possible presence of rionpancreatic endopeptidases in the rat small intestine (15). In addition, it has been observed that after a test meal, a considerable increase in plasma amino acids was observed in 5 human subjects with severe pancreatic insufficiency in whom no measurAbbreviations used in this paper: GA3MNA, glutaryl-trialanine4-methoxy-f:l-naphthylamide; MNA, 4-methoxy-f:l-naphthylamide.

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NEUTRAL ENDOPEPTIDASE IN RAT SMALL INTESTINE

able trypsin activity could be detected (Kim YS, unpublished observation). These data, in addition to the fact that aminopeptidase Nand dipeptidyl aminopeptidase IV from brush-border membrane do not readily hydrolyze intact proteins such as insulin B chain and bovine serum albumin (4), strongly suggest that there may be other enzymes in the brush-border membrane that are capable of hydrolyzing these types of proteins into smaller polypeptides. Neutral endopeptidase is found in a variety of tissues such as kidney and brain, where it is thought to play an important role in the degradation of peptide hormones (16-20). In addition, there have been several reports describing the enzyme in the brush-border membranes of mammalian small intestine (21-23), although its importance as a digestive enzyme is not clear. Therefore, in an effort to begin to elucidate the physiologic significance of neutral endopeptidases in the mucosal cells of the mammalian digestive system, we undertook the following studies.

eth y lenediaminetetraacetic acid-phosphate-buffered saline according to the method of Breimer et al. (25). Twelve incubations were necessary to obtain the final crypt cell fraction. Cells from the 12 incubations were pooled into five representative fractions as follows: (a) villus tip, (b) middle villus, (c) villus base, (d) intermediate or mixed portion , and (e) crypt zone. Each fraction contained - 25 %, 25%, 25%, 15% , and 10% of the isolated cells, respectively. as determined by total protein content. The final pellet of each cell fraction was homogenized. and the brush-border membranes of villus and crypt cells were purified from the pooled fractions by the procedure described previously (25). Brush-border membranes were also prepared from the mucosa of Thiry-Vella loops , 72 h after surgery, as previously described (15).

Materials and Methods Chemicals G1utary I-trialanine-4-met hoxy - f3- na ph thy lamide (GA 3MNA) , trialanine-4-methoxy-f3-naphthylamide, dialani ne-4-methoxy - f3- na phth y lam ide, alanine-4-methoxy - f3naphthylamide (alanine-MNA), 4-methoxy-f3-naphthylamide (MNA)-tosylate, and leucyl-f3-naphthylamide were purchased from Bachem, Torrance, Calif. Phosphoramidone was purchased from Peninsula Laboratories, Belmont, Calif. Porcine kidney aminopeptidase, insulin B chain, amastatin, and other protease inhibitors were obtained from Sigma Chemical Co., St. Louis, Mo. Carrier free Nae 25 I) (20 Ci/mg) was purchased from New England Nuclear, Boston, Mass. All other chemicals used in this study were of reagent grade quality.

Isolation of Brush-Border Membranes Male Wistar rats (Simonsen Lab, Gilroy, Calif.) weighing -300 g were maintained on a standard laboratory diet. Animals were fasted overnight, killed by decapitation, and the small intestine was removed. The intestinal lumen was washed with 2 mM Tris-HCI, 50 mM mannitol buffer, pH 7.1, and the mucosa was obtained by gentle scraping with a glass slide. Brush-border membranes were prepared from the mucosal scrapings by the method described by Kessler et a1. (24) . For some studies, the whole small intestine was removed and divided into six segments of equal length (18 cm). Brush-border membranes were isolated from each segment by the same procedure described above. A gradient of intestinal cells from villus tips to crypt was obtained by serial incubation of excised rat proximal intestine in

1235

Endopeptidase Assay Hydrolysis of insulin B chain .. Insulin B chain was labeled with 125 1 and isolated from excess 1251 by the procedure of Glover et al. (26) . With this method . - 91 % of the radioactivity was precipitated with trichloroacetic acid using the standard enzyme assay procedure. In this assay. enzyme was incubated with 125I_insulin B chain (5.1 mM) in 0.1 M Tris-HCl buffer, pH 7.5 , in a total volume of 0.5 ml. After incubation for 12 min at 37°C the reaction was stopped by the addition of 0.5 ml of 5% bovine serum albumin and 1.0 ml of 15% trichloroacetic acid. After a brief period of centrifugation the amount of radioactivity remaining in the supernatant was determined with a 'Y-scintillation counter. The amount of radioactivity (counts per minute) in the supernatant was corrected for any nonprecipitable radioactivity in the controls and expressed as the percentage of breakdown of 1251-insulin B chain. Enzyme amounts and incubation times were adjusted in each experiment so that the response was on the linear part of the standard curve. A unit of activity is defined as the hydrolysis of 1 nmol of 125I-insulin B chain per minute at 37°C. Hydrolysis of glutary l-trialanine-4-methox y-f3naphthylamide. Endopeptidase activity was also monitored by following the release of fluorogenic MNA from GA3MNA (200 /J-M) after incubation at 37°C for 2 h in 0.05 M Tris-HCl, pH 7.5. The reaction was terminated by placing the tube in a boiling water bath for 2 min . The amount of MNA released was determined by using a spectrofluorometer with excitation and emission wavelengths of 315 nm and 420 nm. respectively. A standard curve with MNA-tosylate was used to convert the relative fluorescence into nanomoles of substrate hydrolyzed. A unit of enzyme activity was defined as the hydrolysis of 1 nmol of substrate per minute at 37°C.

Assay of Other Enzymes and Protein Aminopeptidase N (27), sucrase (28), trypsin (29), chymotrypsin (29). and carboxypeptidase A (30) and B (31) were assayed by previously described procedures. Protein was measured by the modified method of Lowry et a1. (32).

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SONG ET AL.

Assay of the Hydrolytic Products of Glutaryl-Trialanine-4-Methoxy-f3Naphthylamide The concentration of free MNA was determined flu oro metrically as described above, whereas the amounts of alanine-, dialanine-, and trialanine-MNA were monitored with a Beckman model 119CL amino acid analyzer (Beckman Instruments, Inc., Palo Alto, Calif.). Nonfluorogenic alanine, dialanine, and trialanine derivatives were determined indirectly as follows. After incubation with enzyme, the reaction mixture was lyophilized and redissolved in one-tenth the original volume of water. Chromatography of alanine, alanine-MNA, dialanine-MNA, trialanine-MNA, MNA-tosylate, and hydrolytic products was carried out on thin-layer silica gel plates using a butanol/acetic acid/water (4: 1: 1, vol/vol/vol) solvent system. Products were detected with either a ninhydrin spray or ultraviolet light. Areas containing MNA derivatives were compared with known standards, scraped off the thin-layer chromatography plates, dissolved in distilled water, and hydrolyzed to the free fluorogenic MNA product by addition of porcine kidney aminopeptidase N. The amount of fluorescence was used as a measure of the amount of initial hydrolytic product formed. With this indirect method, 64% of a known amount of alanine-MNA standard applied to a silica gel plate could be quantitatively recovered.

Determination of pH Optimum, Thermostability, Kinetic Constants, and the Effect of Inhibitors The pH optimum of endopeptidase was determined using 0.05 M of 0.1 M Tris-HCI buffer for both GA3MNA and 125I-insulin B chain in the range of pH 6.0 to 9.0. Heat stability was determined by means of 20-, 40-, and 60-min preincubations of brush-border membrane at temperatures ranging from 40° to 70°C. Kinetic constants were determined for both 125I-insulin B chain (1.28-25.56 pM) and GA3MNA (20-300 pM) using Lineweaver-Burke plots. The effect of various proteinase inhibitors on the neutral endopeptidase from rat intestinal brush-border membranes was determined after preincubation of the enzyme preparation with the various inhibitors at room temperature for 30 min.

Solubilization of Brush-Border Membrane Neutral Endopeptidase Triton X-I00 (5%, vol/vol) was added to purified brush-border membrane fractions, incubated on ice for 1 h, and briefly sonicated for 10 s. The preparation was then centrifuged at 27,000 g for 30 min, and the supernatant was used for enzyme assays and electrophoretic analysis. In some cases Triton X-I00 (1%) solubilized brush-border membranes were incubated with a mixture of papain (0.4 mg/ml), bromelain (1.5 mg/ml), and cysteine hydrochloride (0.2 mg/ml) at 37°C for 1 h (23) and then centrifuged at 27,000 g for 30 min. The supernatant was carefully removed and used in the electrophoretic studies.

GASTROENTEROLOGY Vol. 91, No.5

Electrophoretic Studies and Molecular Characterization Disc gel electrophoresis of solubilized brushborder membranes and elution of enzyme from gel slices were carried out as described previously (33). Endopeptidase activity was monitored in the eluates by addition of excess purified porcine kidney aminopeptidase to the assay mixture. For the determination of molecular weight, protein standards (transferrin, catalase, ferritin) were electrophoresed in 5%, 7%, and 9% gels and stained with Coomassie Blue. Solubilized brush-border membranes were electrophoresed in identical gels, and endopeptidase activity was measured in eluates from gel slices. The slope of the line obtained from Ferguson plots was used in the determination of the molecular weight, as described by Hedrick and Smith (34).

Intestinal Perfusion of Glutaryl-Trialanine-4Methoxy-f3-Naphthylamide Male Wi star rats weighing ~250 g and maintained on a standard laboratory diet were used in this study. All experiments were conducted with animals that had been fasted overnight. A steady-state perfusion technique described in detail in previous communications was used (35). Test solutions contained GA3MNA (2 mM) in the presence or absence of phosphoramidone (0.15 mM) with [14C]polyethylene glycol as a nonabsorbable marker. Test solutions were in phosphate-buffered saline (10 mM phosphate, pH 7.2), and the sodium chloride concentration was adjusted to make them isoosmotic. Solutions were maintained at 37°C and perfused into a 20-cm jejunal segment beginning 5 cm distal to the ligament of Treitz. A flow rate of 19 ml/h was maintained with a Harvard (model 2681) infusion apparatus (Harvard Apparatus Co., Inc., South Natick, Mass.). After a 30-min equilibration period to achieve steady-state conditions, three consecutive 10-min samples were collected and immediately frozen. At the end of the experiment, the perfused segment was removed and the mucosa was scraped, weighed, and frozen until further use. Products of GA3MNA hydrolysis were monitored in the perfusates by thin-layer chromatography. The concentration of free alanine was measured by amino acid analysis, and the amount of MNA was determined fluorometrically. The [14 C]polyethylene glycol content of test solutions and perfusates was measured with a Beckman scintillation counter (Beckman Instruments, Fullerton, Calif.). Luminal appearance rates of alanine and MNA were calculated as described previously (35).

Results Isolation and Solubilization of Brush-Border Membranes

Levels of sucrase and aminopeptidase N activity were used as a criteria for the purity of brushborder membranes. As shown in Table 1, the specific

November 1986

NEUTRAL ENDOPEPTIDASE IN RAT SMALL INTESTINE

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Table 1. Activity of Neutral Endopeptidase in Brush-Border Membranes of Rat Small Intestine Specific enzyme activityQ (Ulmg protein)b Enzyme Neutral endopeptidase GA3MNA 12sI-insulin B chain Aminopeptidase N Leu-I3-Nap Sucrase Sucrose

Recovery in BBM

Enrichment in BBM

Homogenate

BBM

(%)

(%)

1.39 :+: 0.23 0.72 :+: 0.17

19.29 :+: 4.78 11.73 :+: 3.2

15.4 18.2

13.9 16.4

96.54 :+: 6.57

1138.8 :+: 222.23

13.1

11.8

14.8

13 .4

44 .2 :+: 18.32

592 .2 :+: 152

BBM. brush-border membrane; GA3MNA. glutaryl-trialanine-4-methoxy-f3-naphthylamide. a Mean value (:+: SE) of five different experiments. b A unit (U) of enzyme activity is defined as the hydrolysis of 1 nmol of substrate per minute at 37°c'

activities of sucrase and aminopeptidase N in the brush-border membranes were increased 12-13-fold over that in the homogenate. By comparison. the enzyme activity hydrolyzing 125I-insulin B chain and GA3MNA was enriched 15- and 14-fold. respectively, in the brush-border membrane fraction. The mucosal homogenate and brush-border membranes obtained from intestinal Thiry-Vella loops had levels of enzyme activity toward GA3MNA that were the same as those from normal intestine. The supernatant (100,000 g) of Triton X-l00 solubilized brushborder membranes contained 89%-94% of the original endopeptidase activity. Assay of Hydrolytic Products Using Glutaryl-Trialanine-4-Methoxy-f3Naphthylamide as Substrate To define the precise mechanism of hydrolysis of GA3MNA by brush-border membrane enzymes, hydrolytic products were separated and analyzed by thin-layer chromatography (Figure 1). Alanine, alanine-MNA, dialanine-MNA, and trialanine-MNA standards were well resolved from each other; however, alanine migrated very close to GA3MNA. These two compounds could be distinguished from one another, however, on the basis of their color reaction with ninhydrin. When GA3MNA was incubated with brush-border membranes, flu orogenic MNA was one of the major products (Figure 1). When GA3MNA was incubated with brush-border membranes pretreated with amastatin, a specific inhibitor of aminopeptidase, alanine-MNA was a major hydrolytic product, whereas dialanine-, and trialanine-MNA were not detected. Figure 2 shows the timedependent release of alanine, MNA, and alanineMNA during hydrolysis of GA3MNA by brushborder membranes. As seen in Figure 2B for untreated brush-border membranes, alanine and MNA were produced in equal concentrations linearly for periods of up to 4 h. In addition, the concentration of alanine-MNA produced by amasta-

tin-pretreated brush-border membranes (Figure 2A) was similar to that of alanine and MNA produced by brush-border membranes in the absence of inhibitor. The appearance of alanine-MNA under these conditions was also linear with incubation time. Kinetic Constants, pH Optimum, and Thermostability Using 125I-insulin B chain and GA3MNA as substrates, the Michaelis-Menton constant was estimated to be 36 J.LM and 114 pM, respectively. Maximum velocities were 130 nmol/min· mg brushSOLVENT FRONT

0

4

INA

. 4 AII.INA

0

•

• 4AI1 2'MNA .4AII3'MNA

.4 ORIGIN

CD

Ala

2

Figure 1. Thin-layer chromatograp hy of the hydrolytic products of glutaryl-trialanine-4-methoxy-f3-naphthylamid e (GA3MNA). Peptide standards and products were detected by means of a ninhydrin spray. Fluorescent 4-methoxy-f3-naphthylamine (MNA) was visualized under ultraviolet illumination . Lane 1. GA3MNA substrate. Lane 2. Potential products of GA3MNA hydrol ysis. Lane 3. Hydrolytic products produced by purified brush-border membra nes. Lane 4. Hydrolytic products produced by amastatin (40 JLg/ml)-pretreated brushborder membranes.

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GASTROENTEROLOGY Vo!' 91, No.5

SONG ET AL.

12

Table 2. Effect of Inhibitors on Proteolytic Activities in Brush-Border Membranes

8

A .f'.

.../ .""

Percentage inhibition b

c: 80

o

Inhibitors a

...............

c:

Q)

g 40 o

()

........

o

.....

f',"""

.·_···6

2

3

4

incubation time (H)

Figure 2. Quantitative assay of the hydrolytic products of GA3MNA. Assay of the individual products is described in Materials and Methods. Hydrolytic products were produced by amastatin-pretreated brush-border membranes (A) and purified brush-border membranes (B). Alanine (e-e), alanine-MNA (6---6), MNA (0--0).

border protein and 41 nmol/min . mg brush-border protein, respectively. Hydrolysis of 1251-insulin B chain and GA3MNA by brush-border membranes had a relatively sharp pH optimum of 7.2-7.5. Both activities were relatively stable to prolonged (60 min) heat treatment at 50°C. Enzyme activity was rapidly lost, however, at 60°C. Effect of Various Inhibitors on Enzyme Activity Hydrolysis of 1251-insulin B chain and GA3MNA were both strongly inhibited by metal chelating agents and thiol reagents (Table 2). Phosphoramidone (30 J-LM) inhibited the release of MNA from GA3MNA by 83% and the release of trichloroacetic acid-soluble radioactivity from 125 1_ insulin B chain by 44%. Amastatin, a specific inhibitor of aminopeptidase (36), inhibited the appearance of MNA by 98% and the hydrolysis of 1251-insulin B chain by 32%. Inhibitors of serine and cysteine type endopeptidases, trypsinlike enzymes, carboxypeptidase, and dipeptidyl carboxypeptidase had no significant effect on neutral endopeptidase activity. Molecular Characterization

Brush-border membranes solubilized in Triton X-l00 (5% vol/vol) displayed a single peak of GA3MNA hydrolyzing activity after electrophoresis in polyacrylamide gels (not shown). Using protein standards and Ferguson plots, the molecular weight of this component was estimated to be 265 kilodaltons. Similarly, a single peak of 1251-insulin B chain hydrolyzing activity was observed, which was

None EDTA EGTA 1.10-Phenanthroline Dithiothreitol Phenyl methylsulphonyl fluoride Iodoacetate 4-Hydroxymercuribenzoate Soybean trypsin inhibitor Benzamidine Amastatin Phosphoramidone Hydrocinamic acid Captopril Pepstatin Antipain Leupeptin

Concentration

Insulin B chain GA3MNA Leu-f3-Nap

lmM 1 mM 1 mM 2mM 2mM

0 67.9 60.4 93.1 68.4 0

lmM lmM

0 0

0 0

0 0

100 JLg/ml

0

0

0

1 mM 40 JLg/ml

0 31.9 43.6 0 0 0 0 0

0 97.8 82.7 0 0 0 0 0

0 98 0 0 0 0 0 0

30 JLM

2mM 10 JLM

27 JLg/ml 42 JLg/ml 26 JLg/ml

0 100 100 100 100 0

0 56.8 63.1 93.7 63.1 0

EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycolbis(f3-aminoethylether)-N,N' -tetraacetic acid. a Brush-border membranes were preincubated with various inhibitors at the concentration shown for 30 min at room temperature before assay. b Results given are the mean values obtained in four separate experiments.

very slightly resolved from the GA3MNA hydrolyzing peak. Its molecular weight was estimated to be ~360 kilodaltons. Brush-border membranes that were solubilized with Triton X-l00 (1%) and treated with papain and bromelain gave a different electrophoretic profile (Figure 3). Two peaks of activity were observed that hydrolyzed both GA3MNA and 1251-insulin B chain. The faster migrating form had a molecular weight of 100 kilodaltons and the slower peak had a molecular weight of 565 kilodaltons. Both of these peaks were only partially inhibited by phosphoramidone. A third peak of activity completely resolved from the other two, hydrolyzed 1251-insulin B chain, but was inactive with GA3MNA. This peak of activity was completely inhibited by phosphoramidone (30 J-LM) and had an estimated molecular weight of 390 kilodaltons. Papain and bromelain activity were localized in gel slices No.1 and No. 23, respectively. Regional and Cellular Distribution of BrushBorder Membrane Endopeptidase

Enzyme activities in brush-border membranes from different regions along the longitudinal axis of rat small intestine are shown in Figure 4. Neutral

NEUTRAL ENDOPEPTIDASE IN RAT SMALL INTESTINE

November 1986

1239

I

~!

~1200

30

-,..=

~

~ 800

20

•

> ,....

::; 400

o

10

c

N

Figure 3. Electrophoretic mobility of solubilized brush-border membrane neutral endopeptidase. Triton X-100 solubilized brush-border membranes were treated with papain and bromelain and centrifuged at 27,000 g for 30 min. An aliquot of the supernatant fraction was electrophoresed under nondenaturing conditions in 7% acrylamide gels, as described. Eluates from gel slices were assayed with GA3MNA (0 - -0 ) or 125I_insulin B chain (e-e).

endopeptidase activity was maximal in the middle region of the small intestine with the lowest values found in the distal segment, a pattern similar to that of aminopeptidase N. Figure 5 shows the distribution of neutral endopeptidase activity along the villus-crypt cell axis in proximal jejunum. The profile of neutral endopeptidase was similar to that of aminopeptidase N, with the enzyme levels in villus cells being 3.5-fold to fivefold higher than that of the crypt cells .

~1

I

'" 120 :::

30

,..'"

20

800

VI

Perfusion Studies When GA3MNA (2 mM) was perfused through a 20-cm segment of rat jejunum, free alanine and MNA were measured in the perfusates as the primary hydrolytic products. The mean appearance rates for alanine and MNA in the perfusates were 900 and 367 nmol/60 min· g mucosa, respectively (Figure 6). Phosphoramidone strongly inhibited the appearance of both products by 61 %-63%. The difference in appearance rates between alanine and MNA was attributed to the fact that MNA (0.2 mM) was efficiently absorbed when perfused through intesti-

A

10

1M

Figure 5. Distribution of neutral endopeptidase in brush-border mJ'lmbranes along the villus-crypt axis in the small intestine. Isolated cells were pooled into five fractions representing the following: V{, villus tip; V2, middle villus; V3, villus base; IM, intermediate or mixed region, and C, crypt zone, as described. Neutral endopeptidase and aminopeptidase N activities were assayed using GA3MNA (e-e) or leucyl-f3-naphthylamide (Li- -Li), respectively. Data are the mean value of four experiments.

w ~ c a:: ::; 400

V3

VZ

VILLUS·CRYPT HIS

W

(J

z

c a:: c w

Q. Q.

c

B

•• 0

~

1.0

:sE

CI

0

....

CI

...

'0 E

0.5

INTESTINAL SEGMENTS

Figure 4. Distribution of neutral endopeptidase in the brushborder membrane along the longitudinal axis of the small intestine. Rat small intestine was divided into six equal segments [from A (proximal) to F (distal) , 18 cm each]. Endopeptidase activity was monitored with GA3MNA (e--e). Aminopeptidase N activity was assayed using leucyl-f3-naphthylamide (Li--Li) as substrate. Data are the mean value of six experiments.

Figure 6. Luminal appearance rates of alanine and MNA during intestinal perfusion of GA3MNA. GA3MNA (2 mM) was perfused through proximal intestinal segments (20 cm) in the presence (closed boxes) or absence (open boxes) of phosphoramidone (0.15 mM). Perfusates were analyzed for alanine (A) and MNA (B). Each value represents the mean appearance rate :±: SEM obtained from seven experiments. *p < 0 .005 ; **p < 0.001.

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SONG ET AL.

nal segments. After control perfusions of GA3MNA (2 mM) or phosphoramidone (0.15 mM), no changes were evident in the microscopic structure of the intestinal mucosa, and phosphoramidone had no effect on the intestinal absorption rates of alanine (2 mM). Trypsin, chymotrypsin, and carboxypeptidase A and B activities were not detected in either perfusates or cellular homogenates prepared from mucosal scrapings of perfused intestinal segments.

Discussion Membrane-bound neutral metalloendopeptidases have been described and characterized in a variety of mammalian tissues. Among these are the brush-border membrane of kidney tubules, the synaptic membrane of the brain, and the brush-border membrane of intestinal epithelial cells (37). The role of these enzymes in the kidney and central nervous system is thought to primarily involve the degradation of peptide hormones, whereas the intestinal enzyme has been hypothesized, but never shown, to playa role in protein digestion (38). Previous studies from our laboratory have suggested that intestinal endopeptidases may be of importance during the digestion of protein in rats with experimentally induced pancreatic insufficiency (15). In this report we looked for the presence of these types of enzymes associated with rat intestinal enterocytes in order to more accurately assess their possible physiologic role in the digestion process. As this study demonstrates, enzymes capable of hydrolyzing endopeptidase-type substrates such as GA3MNA and 1251-insulin B chain were found in rat intestinal mucosal cells, were shown to be localized to the brush-border membrane fraction, and were not of pancreatic origin. The fact that the hydrolysis of GA3MNA and 1251-insulin B chain is mediated by neutral metalloendopeptidases in purified rat intestinal brushborder membranes is supported by the following observations. Both substrates are specific for endopeptidase-type enzymes and have been successfully used in studies by other workers (16-20,39). The pH optimum for hydrolysis of both substrates is in the region of 7.0-7.5, and the enzymatic activity is sensitive to metal chelating agents and sulfhydryl reagents. In addition, enzymatic activity is strongly inhibited by phosphoramidone, a specific inhibitor of known neutral metalloendopeptidase-type enzymes (40). When compared with other peptidase enzymes in the brush-border membrane, the levels of endopeptidase activity were quite low (Table 1), suggesting that it is not a major membrane constituent like aminopeptidase N (9) or sucrase-isomaltase

GASTROENTEROLOGY Vol. 91. No.5

(41). This is in contrast to the situation in the kidney microvillus membrane, where endopeptidase levels are reportedly much higher (21,38). Our electrophoretic studies indicate that there may be at least two different endopeptidase enzymes in the rat intestinal brush-border membrane. One of these hydrolyzes both GA3MNA and 1251-insulin B chain, whereas the other hydrolyzes only 125 1_ insulin B chain. The detergent form of the enzyme hydrolyzing GA3MNA has a molecular weight of 265 kilodaltons, whereas the proteinase form (Figure 3) gave two peaks of 100 and 565 kilodaltons. These results are very similar to those of Danielsen et al. (21), who described a 100-kilodalton monomeric form and a high molecular weight aggregate after proteinase treatment of partially purified endopeptidase from pig intestine. Their studies also indicate that the detergent-solubilized enzyme exists as a dimer of 320 kilodaltons. Our results differ from theirs, however, in that the enzyme described here is only partially inhibited by phosphoramidone. The endopeptidase that hydrolyzes 1251-insulin B chain but not GA3MNA (Figure 3) appears to be a separate enzyme based on its substrate specificity, sensitivity to phosphoramidone, and molecular weight. Although a second endopeptidase has been described and characterized in mouse kidney (42), it appears to be different from the 1251-insulin B chain hydrolyzing enzyme described here. The possibility of multiple endopeptidases associated with the rat intestinal brush-border membrane may also explain the discrepancy between our characterization studies and an earlier report dealing with a similar enzyme in rat intestine (23). These differences include the pH optimum, MichaelisMenton constant, thermostability, and sensitivity to various classes of inhibitors, suggesting that these investigators may have been dealing with a different enzyme. The regional distribution profile of endopeptidase activity in the rat small intestine was very similar to that of aminopeptidase N, with maximal activity in the midregion decreasing toward the distal end. Endopeptidase activity was maximally expressed in the differentiated villus cells, as has been described for other brush-border membrane enzymes. Hydrolysis of the two peptide substrates used in this study is a two-step process requiring the active participation of aminopeptidase N. As the data show, when amastatin was used to specifically inhibit aminopeptidase N, the appearance of the final hydrolytic products of both GA3MNA and 1251_ insulin B chain was inhibited even though endopeptidase activity itself was unaffected by amastatin. The results indicate that in the case of GA3MNA, a

November 1986

single peptide bond is cleaved by brush-border membrane endopeptidase to release alanine-MNA. Aminopeptidase N then cleaves this product to free alanine and fluorescent MNA. Thus, stoichiometric amounts of both of these products are observed when brush-border membranes are incubated with GA3MNA for periods of up to 4 h (Figure 2). Although a detailed analysis of the hydrolysis of 1251_ insulin B chain was not carried out, the data strongly suggest that both endopeptidase and aminopeptidase N actively participate in its hydrolysis. Similarly, analysis of the hydrolytic products after in vivo perfusion of Ga3MNA in the presence and absence of phosphoramidone indicated that endopeptidase and aminopeptidase were responsible for hydrolysis of this substrate. Although it is difficult to compare these results with previous studies (35), luminal appearance rates of alanine were 20% of those observed for leucine when the aminopeptidase N substrate Leu-Gly-Gly-Gly was perfused through intestinal segments at comparable concentrations. As aminopeptidase is a major constituent of the intestinal brush-border membrane, the implication is that these membrane endopeptidases may playa potentially important physiologic role in protein digestion. The peptidases of the intestinal brush-border membrane that have been studied to date are generally incapable of hydrolyzing large polypeptides or proteins. Their main role in the digestion process is to break down the small oligopeptides present in the intestinal lumen, which are the products of gastric and pancreatic proteases. However, studies have shown that assimilation of dietary protein can occur in animals after pancreatic duct ligation or pancreatic resection (43,44). A similar situation has been described in humans after removal of the pancreas and most of the stomach (45). Coupled with previous studies from our laboratory (15) and the results presented here, these observations suggest that there may be an important alternative pathway for the hydrolysis of dietary protein in lieu of pancreatic enzymes. In support of this hypothesis, the present study indicates that it may be possible to invoke a mechanism whereby endopeptidases and other proteolytic enzymes of the intestinal brush-border membrane work in concert to degrade relatively large proteins and polypeptides. This process may be of nutritional and physiologic importance to individuals with pancreatic insufficiency. Thus, the neutral endopeptidases of the intestinal microvillus membrane represent a new class of important enzymes worthy of intensive study.

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References 1. Fujuta M, Parsons DS, Wojnarowska F. Oligopeptidases of brush border membranes of rat small intestinal mucosal cells. J Physiol (Lond) 1972;227:337-94. 2. Wojnarowska F, Gray GM. Intestinal surface peptide hydrolases: identification and characterization of these enzymes from rat brush border. Biochim Biophys Acta 1975;403: 147-60.

3. Kim YS, Brophy EJ. Rat intestinal brush border membrane peptidase. 1. Solubilization, purification and physicochemical properties of two different forms of the enzymes. J BioI Chern 1976;251:3199-205. 4. Kim YS, Brophy EJ, Nicholson JA. Rat intestinal brush border peptidase. 2. Enzymatic properties, immunochemistry and interactions with lectins of two different forms of the enzyme. J BioI Chern 1976;251:3206-12. 5. Shoaf C, Berko RM, Heizer WD. Isolation and characterization of four peptide hydrolases from the brush border of rat intestinal mucosa. Biochim Biophys Acta 1976;445:684-719. 6. Gray GM, Santiago NA. Intestinal surface aminooligopeptidase. I. Isolation of two weight isomers and their subunits from rat brush border. J BioI Chern 1977;252:4922-8. 7. Kania RK, Santiago NA, Gray GM. Intestinal surface aminooligopeptidases. II. Substrate kinetics and topography of the active site. J BioI Chern 1977;252:4929-34. 8. Danielsen EM, Sjostrom H, Noren 0, Dabelsteen F. Immunoelectrophoretic studies on pig intestinal brush border proteins. Biochim Biophys Acta 1977;4~4:332-42. 9. Benajiba A, Maroux S. Purification and characterization of an aminopeptidase A from hog intestinal brush border membrane. Eur J Biochem 1980;107:381-8. 10. Auricchio S, Greco L, DeVizia B, Buonocore V. Dipeptidyl aminopeptidase and carboxypeptidase activities of the brush border of rabbit small intestine. Gastroenterology 1978;75: 1073-9.

11. Erickson RH, Bella AB, Brophy EJ, et al. Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV. Biochim Biophys Acta 1983;756:258-65. 12. Garvey IQ, Hyman PE, Isselbacher KJ. y-Glutamyl transpeptidase of rat intestine: localization and possible role in amino acid transport. Gastroenterology 1976;71:778-85. 13. Reisenauer AM, Krumdieck CL, Halsted CH. Folate conjugase: two separate activities in human intestine. Science 1977;198:196-7. 14. Ward PE, Sheridan MA. Angiotensin converting enzyme of

rat intestinal and vascular surface membrane. Biochim Biophys Acta 1982;716:208-16. 15. Curtis KJ, Gaines HD, Kim YS. Protein digestion and absorption in rats with pancreatic duct occlusion. Gastroenterology 1978;74:1271-6. 16. Kerr MA, Kenny AJ. The purification and specificity of a

17.

18.

19.

20.

neutral endopeptidase from rabbit kidney brush border. Biochem J 1974;137:477-88. Kerr MA, Kenny AJ. The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border. Biochem J 1974;137:489-95. Almenoff J, Orlowski M. Membrane-bound kidney neutral metalloendopeptidase: interaction with synthetic substrates, natural peptides and inhibitors. Biochemistry 1983;22:590-9. Mumford RA, Pierzchala PA, Strauss AW, Zimmerman M. Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. Proc Nat! Acad Sci USA 1981;78:6623-7. Orlowski M, Wilk S. Purification and specificity of a mem-

1242

21.

22.

23 . 24.

25 . 26. 27.

28. 29 . 30. 31. 32. 33.

SONG ET AL.

brane-bound metalloendopeptidase from bovine pituitaries. Biochemistry 1981;20:4942-50. Danielsen EM, Vyas JP, Kenny AJ. A neutral endopeptidase in the microvillar membrane of pig intestine. Biochem J 1980;191:645-8. Slaby I. Fric P, Kasafirek E. Endopeptidase activity of the brush border of human enterocyte. Acta Hepato-Gastroenterol 1978;25 :295-302. Kocna P, Fric P, Slaby J, Kasafirek E. Endopeptidase of the brush border membrane of rat enterocyte. Hoppe-Seyler's Z Physiol Chem 1980;361:1401-12. Kessler M, Acuto 0, Storelli C, et al. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport system. Biochim Biophys Acta 1978;506:136-54. Breimer ME, Hansson GC, Karlsson KA, Leffler H. Glycosphingolipids and the differentiation of small intestinal epithelium. Exp Cell Res 1981;135:1-13. Glover JS, Salter ON, Shepherd BP. A study of some factors that influence the iodination of ox insulin. Biochem J 1967;103:120-8. Bella AM Jr, Erickson RH, Kim YS. Rat intestinal brush border membrane dipeptidyl aminopeptidase IV: kinetic properties and substrate specificities of the purified enzyme. Arch Biochem Biophys 1982;218:156-62. Dahlqvist A. Assay of intestinal disaccharidase. Anal Biochem 1968;27:99-107. Hummel BCW. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem PhysioI1959;37:1393-9. Folk JE, Schirmen EW. The porcine pancreatic carboxypeptidase A system. I Bioi Chem 1963;238:3884-94. Folk JE, Gladner JA. Carboxypeptidase B.IV. Purification and characterization of the porcine enzyme. J Bioi Chem 1960;23 5:2272-7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. I Bioi Chem 1951;193:265-75. Erickson RH, Kim YS. Effect of Triton X-I00 on the electrophoretic mobility of solubilized intestinal brush border mem-

GASTROENTEROLOGY Vol. 91, No . 5

34. 35.

36. 37.

38.

39. 40. 41.

42. 43 . 44. 45.

brane dipeptidyl peptidase IV. Biochim Biophys Acta 1980;614:210--4. Hedrick JL, Smith AJ. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 1968;126:155-64. Chung YC, Silk DBA, Kim YS. Intestinal transport of a tetrapeptide, L-Ieucylglycylglycylglycine in rat intestine in vivo. Clin Sci 1979;57:1-11. Aoyagi T, Tobe H, Kojima F, et al. Amastatin, an inhibitor of aminopeptidase A produced by actinomycetes. J Antibiot 1978;31 :636-8. Gee NS, Bowes MA, Buck p, Kenny AI. An immunoradiometric assay for endopeptidase-24. 11 shows it to be a widely distributed enzyme in pig tissues. Biochem J 1985;228: 119-26. Kenny I. Gee N, Matsas R, Stewart I. Bowes B, Turner A. Endopeptldase-24. 11: a general purpose membrane enzyme for metabolising peptides. In: Khairallah EA, Bond IS, Bird JWC, eds. Intracellular protein catabolism. New York: Alan R. Liss, 1985:175-84. Kenny AI. Fulcher IS, Ridgwell K, Ingram J. Microvillar membrane neutral endopeptidases. Acta Bioi Med Germ 1981 ;40:1465-71. Suda H, Aoyagi T, Takeuchi T, Umezawa H. A thermolysin inhibitor produced by streptomyces; phosphoramidone. I Antibiot 1973;26:621-3. Kenny AI. Maroux S. Topology of microvillar membrane hydrolases of kidney and intestine. Physiol Rev 1982;62: 91-128. Beynon RI. Shannon JD, Bond JS. Purification and characterization of a metallo-endopeptidase from mouse kidney. Biochem J 1981;199:591-8. Uram JA, Friedman L, Kline OL. Relation of pancreatic exocrine to nutrition of the rat. Am J Physiol 1960;199: 387-94. Anderson OM, Ash RW. The effect of ligating the pancreatic duct on digestion in the pig. Proc Nutr Soc 1971;30:34A-5A. Crane CWo Studies on the absorption of 15N labelled yeast protein in normal subjects and patients with malabsorption. In: Munro HN, ed. The role of the gastrointestinal tract in protein metabolism. Philadelphia: FA Davis, 1964:333--47. '