Clinical Science (1979) 57, 1-1 1

Intestinal transport of a tetrapeptide, L-leucylglycylglycylglycine,in rat small intestine in vivo

Y. C . C H U N G , D. B. A. SIL K

AND

Y. S. K I M

Gastrointestinal Research Laboratory. Veterans AdministrationHospital, Son Francisco. California.and the Department of Medicine, Universityof Cal~ornia,School of Medicine, San Francisco, California, U S A .

(Received 6 June 1978; accepted 20 February 1979)

brush-border aminopeptidase activity by 85% in vitro, failed to block substantially net Leu absorption from Leu-Gly and Leu-Gly-Gly-Gly. 5. The data presented suggest that, although some of the Leu from the tetrapeptide, Leu-GlyGly-Gly, may be hydrolysed before transport, nearly 50% of the tetrapeptide appears to be transported intact. Although Leu-Gly, Leu-GlyGly and Gly-Pro seem to share a common transport mechanism, the system used for intact Leu-Gly-Gly-Gly absorption seems to be distinct. However, the present study does not exclude the possibility that binding of the tetrapeptide to the brush-border aminopeptidase alters the atlinity of Leu for the amino acid carrier, and therefore further studies are necessary before firm conc1usior)s can be made on the general mechanism of tetrapeptide transport.

Summary 1. The intestinal transport mechanism for the tetrapeptide L-leucylglycylglycylglycine, Leu-GlyGly-Gly, and its relation to the transport of free Leu, Leu-Gly and Leu-Gly-Gly were investigated Lt vivo by means of jejunal perfusion in rats. 2. The rates of net Leu absorption from peptides (Leu-Gly and Leu-Gly-Gly-Gly) were significantly greater than those from the free amino acid mixtures when the test solutions were perfused at a concentration of 15 mmol/l. 3. Net Leu absorption rates from Leu-Gly (10 ymol/ml) and Leu-Gly-Gly (10 ,vmol/ml) were extensively inhibited (84% and 68% respectively) by Gly-Pro at 100 mmol/l, whereas Gly-Pro had no effect on Leu absorption from Leu-GlyGly-Gly. L-Alanine (Ala, 100 ymol/ml), on the other hand, which completely inhibited Leu absorption during perfusion of free Leu, inhibited Leu uptake from Leu-Gly-Gly-Gly only about 50% at all concentrations studied. Ala had no effect on Leu absorption from Leu-Gly and Leu-Gly-Gly (10 y mollml). 4. Neither Ala at 100 pmol/ml nor Gly-Pro at 100 ymol/ml had any effect on brush-border aminopeptidase activity in vitro, suggesting that the hydrolytic capacity of the intestinal mucosal brush border was unaltered when Ala or Gly-Pro was included in the perfusion mixture. L-Alanyl-fi naphthylamide (20 ymollml), which inhibited

Key words: intestinal transport, L-leucylglycylglycylglycine, peptidase, tetrapeptide. Abbreviations: ANA, L-alanyl-finaphthylamide; PEG, polyethylene glycol. I

Introduction

In the lumen of duodenum and proximal jejunum, proteins and polypeptides are hydrolysed by the action of pancreatic enzymes (Gray 8c Cooper, 1971) yielding mixtures of free amino acids and oligopeptides containing two to six amino acid units (Chen, Rogers 8c Harper, 1962; Adibi 8z Mercer, 1973). The process involved in the absorption of the final products of luminal proteolytis

Correspondence: Dr Young S. Kim, Gastrointestinal Research Laboratory, Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121,

USA. 1

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Y. C. Chung, D. B. A . Silk and Y. S . Kim

are complex. Amino acids are absorbed by at least four group-specific amino acid-transport systems (Saunders & Isselbacher, 1966). Information derived from many studies in vitro and in vivo show that unhydrolysed di- and tri-peptides may also be translocated across the microvillus membrane of the intestinal mucosal cell by carriermediated transport processes (Silk, 1974; Matthews, 1975; Matthews & Adibi, 1976; Adibi, 1976). The cytoplasmic compartment of the mucosal cell contains peptidase activity against diand tri-peptides (Robinson & Shaw, 1960; Josefsson & Sjostrom, 1966; Peters, 1970; Donlon & Fottrell, 1972; Kim, Birtwhistle & Kim, 1972; Heizer, Kerley & Isselbacher, 1972), and since only a few unhydrolysed peptides have been identified in the portal and peripheral circulation during absorption experiments (Prockop & Sjoerdsma, 196 l), most translocated di- and tripeptides are thought to be hydrolysed by cytoplasmic peptide hydrolases to free amino acids (Silk, 1974; Matthews, 1975; Matthews & Adibi, 1976; Adibi, 1976). A second group of peptide hydrolases has been identified in association with the brush-border fraction of the mucosal cell and shown to hydrolyse di-, tri- and higher peptides (Peters, 1970, 1973); Donlon & Fottrell, 1972; Kim et al., 1972; Heizer et al., 1972; Fujita, Parsons & Wojnarowska, 1972; Kim, Kim & Sleisenger, 1974; Kania, Santiago & Gray, 1977). These enzymes are intrinsic constituents of the microvillus membrane (Louvard, Maroux, Rannier & Desnuelle, 1975a) and their catalytic sites are positioned on the luminal side of the membrane (Louvard et al., 1975a; Louvard, Maroux & Desnuelle, 1957b). Characterization of these enzymes has provided support for the original suggestion that there are two major modes of uptake of di- and tri-peptides (Cheng, Navab, Lis, Muller & Matthews, 1971; Matthews, 1971; Bonllin, Crampton, Heading & Pelling, 1973; Silk, 1974): (1) transport of luminal peptides across the microvillus membrane and hydrolysis to free amino acids by cytoplasmic peptidases, and (2) hydrolysis of luminal peptides at the surface of the cell by brush-border peptide hydrolases with release of amino acids into free solution followed by absorption by the group-specific amino acid-transport systems. Currently, it is believed that the functional importance of schemes (1) and (2) is dependent on the affinity of brush-border peptide hydrolases for luminal peptide substrates (Adibi, 1971). The processes involved in the assimilation of tetra-, penta- and hexa-peptides, the other major

products of luminal hydrolysis, are less clear. The following lines of evidence suggest that they are hydrolysed to smaller peptides and free amino acids before transport occurs: (1) the lack of evidence for transport of glycylsarcosylsarcosylsarcosine (Gly-Sar-Sar-Sar), tetraglycine and glycyl-L-leucylglycylglycine (Gly-Leu-Gly-Gly) in an intact form (Matthews & Adibi, 1976; Adibi & Morse, 1977; Smithson & Gray, 1977; Sleisenger, Burston, Dalryrnple, Wilkinson & Matthews, 1976); (2) the lack of cytoplasmic peptidase activity against tetrapeptides (Kim et al., 1974; Schiller, Huang & Heizer, 1977); (3) the localization within the brush border of peptidases capable of hydrolysing tetra- and higher peptides (Fujita et al., 1972; Kim & Brophy, 1976; Kania et al., 1977; Smithson & Gray, 1977). The present study was undertaken to test this hypothesis, namely that a tetrapeptide, L-leucylglycylglycylglycine (Leu-Gly-Gly-Gly) is hydrolysed before uptake of its constituent amino acid residues by brush-border peptide hydrolases whereas signifcant amounts of smaller related peptides, Lleucylglycylglycine (Leu-Gly-Gly) and Lleucylglycine (Leu-Gly), are absorbed intact and hydrolysed by cytoplasmic peptide hydrolases. Methods and materials

Animals Female Wistar rats weighing 240-260 g, maintained on laboratory chow pellets (Ralston Purina, St Louis, MO, U.S.A.) were used throughout the study. All experiments were performed on fed animals since previous studies have shown that keeping rats without food has a deleterious effect on brush-border hydrolase activity (Kim, McCarthy, Lane & Fong, 1973). Material Glycine, Leu-Gly and Leu-Gly-Gly were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Glycyl-L-proline (Gly-Pro) and Lalanyl-8-naphthylamide (ANA) were purchased from Bachem (Marina Del Rey, CA, U.S.A.). LeuGly-Gly-Gly was obtained from Fox Biochemical (Tucson, AZ, U.S.A.). L-Alanine was obtained from Mann Research Laboratories Inc. (New York, N.Y., U.S.A.). The purity of all amino acids and ohgopeptides was checked before study by ion-exchange chromatography. [ 14C1Polyethylene glycol ([14CIPEG, mol. wt. 4000) was obtained from New England Nuclear (Worchester, MA,, U.S.A.).

Intestinal transport of a tetrapeptide Perfusion technique Animals were lightly anaesthetized with diethyl ether and anaesthesia was subsequently maintained by subcutaneous injection of pentobarbital (5 mg/100 g body weight). A tracheostomy was carried out to aid respiration. The peritoneal cavity was opened by a midline incision, and a loop of proximal small intestine 16-20 cm long identified. Each loop was washed with 0.9% sodium chloride solution maintained at 37OC and cannulated proximally (3 cm distal to the ligament of Treitz) and distally with polyethylene catheters, which were secured with silk ligatures. After replacement of the loop in the peritoneal cavity, the test solutions, maintained at 37OC in a water bath, were infused through the proximal catheter at 19 ml/h by use of a Harvard infusion apparatus (model 2681; Millis, MA, U.S.A.). The rectal temperature of each rat was maintained at 37OC throughout each experiment. After a period of 30 rnin to attain a steady state, three 10 rnin collections of the perfusate were made into plastic tubes maintained in a freezing mixture (solid carbon dioxide/ethanol) to prevent further hydrolysis of test peptides by intraluminal peptide hydrolases. Two test solutions were perfused in each animal. At the end of the first perfusion period, the loop was cleared and refilled with the second test solution. ARer a further 30 min equilibration period, three 10 rnin collections were made as before. The sequence of perfusion of different test solutions was always randomized. After collection of intestinal contents, the frozen samples were immersed in a water bath maintained at 95OC for 10 min, to inactivate intraluminal peptide hydrolases, and then stored at -2OOC until analysis. At the end of each perfusion experiment, the perfused segment was removed and the mucosa was scraped from the intestinal wall and weighed.

Experimental design Four series of experiments were performed. Experiment 1. Test solutions contained the peptides Leu-Gly, or Leu-Gly-Gly or Leu-Gly-GlyGly at a concentration of 10 pmol/ml, or an equivalent concentration of the constituent free amino acids. Individual animals were perfused with two test solutions, one containing peptide and the other its respective amino acid mixture. Experiment 2. The effects of Ala on the absorption of Leu during perfusion of the peptides was investigated. Individual animals were perfused with test solutions containing peptide at 1, 2, 5, 10

3

or 15 pmol/ml in the presence and absence of Ala (100 ,umol/ml). Experiment 3. The effect of Gly-Pro on the absorption of Leu during perfusion of the peptides was investigated. Individual animals were perfused with test solutions containing each peptide (10 pmollml) in the presence and absence of Gly-Pro (100 pmollml). Experiment 4. The effect of ANA on the absorption rates of Leu during perfusion of LeuGly or Leu-Gly-Gly-Gly at 1 and 2 mmol/l was investigated. Individual animals were perfused with Leu-Gly and Leu-Gly-Gly-Gly in the presence of ANA (20 pmol/min).

Composition of test solution The test solutions, containing peptides, free amino acids or both, were made iso-osmotic (300 mosmol/l) by adding sodium chloride. Therefore if the test solution contained Ala (100 pmol/ml) or Gly-Pro (100 pmol/ml), there was substantial reduction in the amount of sodium chloride in the perfused solution. This difference in the amount of sodium chloride may have an effect on the net Leu absorption during perfusion of amino acid or peptides. Therefore amino acids (10 pmol/ml) and peptides (10 pmol/ml) in 0.9% or 0.6% sodium chloride solution were examined for net Leu absorption. No statistically significant differences were noted. All test solutions contained the nonabsorbable marker PEG (1 pCi of [l4CIPEG/l) at a concentration of 5 g/l. The pH of all solutions was adjusted to 7 by titration with sodium hydroxide just before perfusion.

Subcellularfractionation Fed animals were lightly anaesthetized with diethyl ether, taken to a cold room maintained at 4 OC and decapitated. Immediately after decapitation, the small intestine distal to the ligament of Treitz was removed. After washing with an iso-osmotic sodium chloride solution (4OC) the mucosa from the proximal 16 cm was scraped and weighed. Mucosal scrapings were homogenized in 14% glycerol solution (14 ml/g of mucosa) in a Potter-Elvehjem homogenizer with eight strokes of a Teflon pestle with a 0~004-0.006 cm clearance, driven by a Con-Torque (Eberbach Corp.,Ann Arbor, MI, U.S.A.), stirred at medium speed, and then passed through two layers of cheesecloth. The homogenate was then centrifuged at 105 OOO g for 1 h. The supernatant was

Y. C . Chung, D. B . A . Silk and Y. S . Kim

4

the enzymes in the two fractions were not due to the effect of the detergent. The electrophoretic technique used was a modification of the method of Davis (1964). After electrophoresis, gels were sliced at 3mm intervals and the slices incubated for 16 h at 37OC in 0.4 ml of Tris/maleate buffer (50 mmol/l), pH 8.0. Portions of the supernant were then assayed for enzyme activity in the absence and presence of p-hydroxymercuribenzoate (0.4 pmoVm1).

designated as the soluble fraction. The pellet containing the brush-border fraction was resuspended in 14% glycerol and centrifuged at 3000 g for 30 min to obtain an enriched brush-border pellet (Kim et al., 1972). This procedure was repeated three times, and the pooled fractions were designated the partially purified brush-border fraction. Preliminary studies showed that more than 96% of the trehalase activity (estimated by the method of Dahlqvist, 1964) in the original homogenate fraction was recovered in the partially purified brush-border fraction. In addition, all peptide hydrolase activity (in the soluble fraction) against Leu-Gly, Leu-Gly-Gly and Leu-Gly-GlyGly was inhibited by p-hydroxymercuribenzoate at a final concentration of 0.4 ,umol/ml, and peptide hydrolase activity in the purified brush-border fractions against the same peptide substrate was not reduced by adding p-hydroxymercuribenzoate to the reaction mixture.

Analytical technique Intestinal contents. During perfusion experiments, net rates of absorption of Leu were measured. Free Leu, Leu-Gly-Gly-Gly, Gly-GlyGly and Gly-Gly in the intestinal samples and test solution were determined by ion-exchange chromatography with a model 120 C Amino Acid Analyzer (Beckman, Palo Alto, CA, U.S.A.). Samples (0.25 ml) were applied to a UR-30 column maintained at 55.5OC and eluted with sodium citrate buffers (0.2 mol/l), pH 3.25 and 4.25, at a flow rate of 68 ml/h. Satisfactory separation of peaks representing Leu, Gly, LeuGly-Gly-Gly, Gly-Gly-Gly, Gly-Gly, Leu-Gly-Gly, Leu-Gly and Ala was achieved (Fig. 1). The [14CIPEG content of test solutions and intestinal contents was measured by a previously reported method (Wingate, Sandberg & Phillips, 1972; Silk, Perrett, Webb & Clark, 1974b). Enzyme assays. For the assay of peptide

Polyacrylamide-gel electrophoresis Before examination of the electrophoretic mobilities of the enzymes in the soluble and partially purified brush-border fractions by polyacrylamide-gel electrophoresis, the membranebound enzymes in the latter fraction were solubilized by using Triton X- 100 as previously described (Kim et al., 1972). Triton X-100was also added to portions of the soluble fraction to ensure that possible differences in the mobilities of

1

0 04

-

1

la'

0 02

E

e

Lo

v

u OI C

2

92

004

0 02

30

60

90

120

Elution time (min)

FIG. 1. Chromatogram showing elution profile of amino acids and peptides in the perfusates (9.25 PI) ( a ) and a standard mixture (0.05 pmol each) ( b)containing substrate and possible hydrolytic products.

Intestinal transport of a tetrapeptide hydrolase activity in the soluble and partially puritled brush-border fractions of intestinal mucosa, a method using the Beckman Amino Acid Analyzer for free Leu, Gly, Gly-Gly and Gly-GlyGly was used. The standard assay mixture contained 375 nmol of peptide substrate and 0.8 pg of protein from the cytosol fraction, or 2-4 ,ug from the brush-border preparation, in KCVborate buffer (50 mmol/l), pH 8.0, in a final volume of 100 pL The reaction mixture was incubated at 37°C for 20 min in a shaking water bath and the reaction stopped by quickly adding 200 pl of 7.5% sulphosalicylic acid. The acidified reaction mixture was dduted as desired by adding sodium citrate (0.2 mol/l, pH 2.2) and stored at -2OOC until analysis on the Amino Acid Analyzer. Under these conditions, the assay was h e a r with respect to time and the amount of enzyme. Control incubations, consisting of either enzyme alone or substrate alone, were carried out. Enzyme samples from the cytosol or brush-border fractions in the absence of substrate did not yield measurable amounts of Leu. Protein was determined by the method of Lowry, Roseborough, Farr & Randall (195 1).

Calculation of results and use of statistics The absorption rate of the amino acid residue Leu during perfusion of peptide solution was calculated by using the formula described previously (Silk et al., 1974b). The net rates of Leu absorption and appearance during perfusion experiments with test solutions containing peptides and amino acids were calculated with the paired and unpaired t-tests when appropriate (Langley, 1968).

5

Experiment 1 There was no significant difference in net rate of Leu absorption during the paired peptide and equirnolar free amino acid perfusion when the concentration of the test solutions was 10 pmollml. The net absorption rates of Leu @mol absorbed min-l g-l of mueosa), when eqnivdent concentrations of the constituent free amino acids were perfused, were: 1.10 f 0.07 (10 mmol of Leu + 10 mmol of Gly), 1.36 k 0.10 (10 mmol of Leu + 20 mmol of Gly) and 1.12 k 0-07 (10 mmol of Leu + 30 mmol of Gly). These data are very similar to those obtained with perfusion of 10 mmol of LeuGly, Leu-Gly-Gly or Leu-Gly-Gly-Gly (Table 1). Similar results were obtained when the concentration of the test solution was Less (Table 1). However, at 15 pmd/ml there was greater absorption (P < 0.05) of Leu from Leu-Gly and LeuGly-Gly-Gly than from the free amino acids (Table 1). The analysis of the perfusam obtained after perfusion with Leu-Gly-Gly-GIy showed the following: Gly, Leu, Gly-Gly, Gly-Gty-Gly, Leu-GlyGly-Gly. No Leu-Gly or Leu-Gly-Gly could be detected.

Experiment 2 The effect of Ala (100 pmollml) on net absorption of Leu during perfusion of the three peptides (10 pmol/ml) and an quimolar amino acid mixture is shown in Tabk 1. The presence of Ala in the free amino acid solution caused a marked reduction, by 95%, in Leu absorption (P< 0.001). Ala also had a significant but less-marked

1. Net absorption rates of leucine during intestinatperfusion in vivo TABLE Each value represents the mean net leucine absorption rate (pnol min-' g-' of mucosa) f SEM obtained from six experiments. The values obtained in the presence of alanine were significantly lower (t-test) than those without it: * P < 0.005; ** P < 0401. N.D., Not determined.

Test solution perfused

Leu + Gly

Net absorption rate ofleucine (units/g of mucosa)

Ala (100 mmolll) added

+

Leu-Gly

-

Leu-Gly-Gly

-

Leu-Gly-Gly-Gly

-

+

+ +

Concn. of amino acids and peptides in test solution 15 mmol/l

10 mmolll

5 mmolll

2 mmoUl

1 mmdll

1.16 f 0.04 0.38 f 0.04. 1.59 f 0.78 1.49 f 0.04

1.10 f 0.07 0.06 f 0.021.16 f 0.03 1.11 f 0.03 1.22 k 0.10 1 . 1 1 f0.03 1.30 f 0.13 0.77 f 0.06*

0.43 f 0.02 0.05 f 0.020.5 1 f 0.02

0 . 15 2 0.03 0.03 f 0.010.22 f 0.01 0-21 f 0.01

0.12 f 0.01 0** 0.12 f 0.01 0.09 & 0.01

N.D. 1.42 f 0.05 0.90 f 0.03.

0.45

0.03

N.D. 0.49 f 0.04 0-27 f 046*

N.D. 0.20 f 0.04 0.10 & 0.01.

N.D. 0.12 f 0.01 0.07 f 0.01'

Y. C. Chung, D. B. A . Silk and Y. S . Kim

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TABLE2. Effect of alanine on net appearance rates of leucine triglycine and diglycine during intestinal perfusion of Leu-Gly-Gly-Gly Each value represents the mean appearance rate (pnol min-l g-' of mucosa) f s m obtained from six experiments. * Net appearance of leucine in the absence and presence of alanine at 100 rnrnol/l was statistically significant at all concentrations studied (P< 0.005, t-test). Concn. of Leu-Gly-Gly-Gly perfused (mmol/l)

Alanine added

TABLE 3. Effect of L-alanyl-/%naphthylamide on leucine absorption during intestinal perfusion of Leu-Gly-Gly-Gly or Leu-Gly Values are means SEM from six experiments. L-Alanyl-p-naphthylamide (ANA) was at 20 pnol/l in the perfused solutions. Percentage inhibition of L-leucine absorption by ANA is shown by values in parentheses.

+

Test solutions perfused

Net absorption rate of leucine

(unit/g of mucosa) Without ANA Leu-Gly ( 1 mmol/l) Leu-Gly (2 mmol/l) Leu-Gly-Gly-Gly ( 1 mmol/l) Leu-Gly-Gly-Gly (2 mmol/l)

Net appearance rate (unit/g of mucosa)

(100 mmol/l)

0.12 f 0 . 0 1 0.22 f 0.01 0.12 f 0.01 0.20 f 0.04

With ANA

Leucine

Triglycine

Diglycine

0.21 f 0.01 0.53 f 0.03. 0.19 f 0.04 0.41 f 0.03. 0.12 f 0.02 0.28 f 0.08. 0.07 f 0.01 0.12 f 0.01' 0.03 f 0 0.07 f 0 .

0.13 f 0.01 0.14 f 0.01 0.12 f 0.02 0.12 f 0.01 0.08 f 0.01 0.08 f 0.01 0.03 f 0 0.04 f 0.01 0.02 f 0 0.02 f 0

0.09 f 0.01 0.10 0.01 0.0s 0 0.05 f 0.01 0.03 f 0 0.03 f 0 0.02 f 0 0.03 f 0 0 0.01 f 0

Gly or Leu-Gly-Gly was detectable during perfusion of Leu-Gly-Gly-Gly. Appearance rates of free Leu increased significantly when each peptide was perfused in the presence of Ala. However, appearance rates of di- and tri-glycine from the tetrapeptide were not affected by Ala. Although not shown in Table 2, Leu appearance rate was faster (P < 0.005) during perfusion of tetrapeptide than during perfusion of Leu-Gly at all concentrations examined.

0~10~0~02(16)

0.23 f 0.02 (0) 0.09 f 0.02 (24) 0.17 f 0.03 (17)

effect on Leu absorption during perfusion of LeuGly-Gly-Gly (P < 0405). In contrast, net Leu absorption rates were similar when the dipeptide and tripeptide were perfused in the absence and presence of Ala. When studies were carried out with various concentrations of amino acids and peptides, similar results were obtained (Table 1). At all concentrations studied Ala caused a marked reduction in the absorption of free Leu; lessmarked reduction was observed in Leu-Gly-GlyGly and the absorption of Leu from Leu-Gly was unaffected. With Leu at 1 pmol/ml virtually no absorption (0.2%) occurred in the presence of Ala ( 100 pmol/ml), but considerable absorption (37%) of Leu occurred during perfusion of Leu-Gly-GlyGly with this inhibitor. Only free Leu and di- and tri-glycine were detected in intestinal contents aspirated during perfusion of Leu-Gly-Gly-Gly (Table 2). No Leu-

*

Experiment 3 The results of competition experiments with GlyPro are shown in Fig. 2. Although Gly-Pro markedly inhibited net uptake of Leu from the dipeptide (73.7%, P < 0.01) and tripeptide (68.296, P < 0.01), Leu absorption rates from equimolar mixtures of amino acids and the tetrapeptide were unaffected by Gly-Pro (100 pmollml).

Experiment 4 Leu absorption rates during the perfusion of test solutions containing Leu-Gly or Leu-Gly-GlyGly at 1 and 2 pmol/ml with ANA (20 pmol/ml) are shown in Table 3. Although ANA (20 pmol/ml) significantly inhibited Leu hydrolysis from L-leucyl peptides by the partially purified brush-border aminopeptidase in uitro (Table 4), it did not affect the absorption rates of Leu to any great extent during perfusion of Leu-Gly or Leu-Gly-Gly-Gly. Some free Ala could be detected in the perfusate, indicating that ANA was hydrolysed by the brushborder aminopeptidase.

Intestinal transport of a tetrapeptide Subcellular distribution of peptidase activity A homogenate of jejunal mucosa contained peptidase activity against all three Leu-containing peptides (Table 5). Although not shown in Table 5 , subcellular fractionation studies showed that nearly all the trehalase activity (96%) was associated with the partially purified brush-border fraction, with only a minimal activity (1%) in the soluble fraction. In contrast, activity against Leu-Gly and Leu-GlyGly as well as Leu-Gly-Gly-Gly was located in both the soluble and partially purified brush-border fractions. Activities against Leu-Gly (8 1.2%) and Leu-Gly-Gly (72.8%) were predominantly localized in the soluble fraction. By comparison, only 52.3% of the activity against Leu-Gly-GlyGly resided in this fraction. The major hydrolysis products of Leu-Gly-Gly and Leu-Gly-Gly-Gly substrates after incubation with either subcellular fraction were Leu and Gly-Gly or Gly-Gly-Gly. No Leu-Gly or Leu-Gly-Gly was detectable during incubation of Leu-Gly-Gly-Gly with either subcellular fraction. Peptidases in the soluble and partially purified brush-border fractions active against the three LeuTABLE4. Effect of L-ahnyl-B-naphthylamide on brushborder peptidase activity with Leu-Gly-Gly-Gly as substrate Enzyme activity is expressed as pmol of leucine released min-I mg-I of protein. Values are means f SEM from

containing peptides had different mobilities when subjected to polyacrylamide-gel electrophoresis (Fig. 3a and 3b). Solubilized peptidases in the partially purified brush-border fraction migrated as two bands (Fig. 3a). Peptidase activity in either band was greater against Leu-Gly-Gly than against Leu-Gly or Leu-Gly-Gly-Gly. Peptidases in the soluble fraction also migrated as two bands (Fig. 3b). In contrast to the enzyme in the partially purified brush-border fraction, the relative activities towards the three substrates varied in each peak. Activity in the band which migrated faster was greater against Leu-Gly-Gly than the other two substrates, whereas in the band which moved more slowly, activity was greater against Leu-Gly.

Eflects of Ala and Gly-Pro on brush-border peptidase activity To investigate the possible effects of Ala and Gly-Pro on brush-border peptidase activity, assays of enzyme activity in vitro in the partially purified brush-border fraction with Leu-Gly, Leu-Gly-Gly and Leu-Gly-Gly-Gly (10 ,umol/ml) as substrates .N.S P < O 01

Without ANA

10 4 2 1

0.87 f 0.06 0.71 f 0.02 0.66 f 0.01 0.55 f 0.01

With ANA 0.31 f 0.05 0.18 f 0.02 0.15 f 0.02 0.09 f 0.03

(60) (75) (78) (86)

P 4 0 01

I,1,

Enzyme activity (unithg of protein)

(mmolA)

1-

N.S.

six experiments. L-Alanyl-pnaphthylamide (ANA) was at 20 pmol/l. Percentage inhibition of peptidase activity by ANA is shown by values in parentheses. Concn. of substrate

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FIG.^. L-Leucine absorption rate (pmol min-l g-I of mucosa) during intestinal perfusion 6f Leu-Gly (10 mmol/l), Leu-Gly-Gly (10 mmol/l), Leu-Gly-Gly-Gly (10 mmol/l) or a mixture of L-leucine and glycine each at 10 mmol/l. Absorption rates in the presence of Gly-Pro at 100 mmol/l (hatched bars) or in the absence of GlyPro (open bars) are shown. N.S., Not significant.

TABLE5 . Subcellular localization ofpeptidase activity in rat jejunal mucosa Enzyme activity is expressed as pmol of leucine released min-' g-a of wet mucosa. Values are means f SEM from six experiments. Percentage of peptidase activity in the homogenateis shown by values in parentheses. Subcellular fraction

Homogenate Soluble fraction Partially purified brush-border fraction

Enzyme activity against peptides (units/g of mucosa) Leu-Gly

Le~-Gly-Gly

Le~-Gly-Gly-Gly

28.75 f 3.12 23.31 1.91 (81)

94.25 f 4.65 68.62 f 5.15 (73)

19.41 f 0.85 10.15 f 1.05 (52)

4.32 f 0.40(152

13.74 f 1.42 (15)

5.85 f 0.65 (30)

8 l5

1

Y. C. Chung, D.B. A . Silk and Y. S. Kim ‘a’

e .

Leu-Glv

0-0 Leu-Glv-Glv

10

A-A Leu-Glv-Gly-Gly

20

0

2

4

6

8

1

0

Distance from top of gel (cm)

FIG.3. Polyacrylamide-gel electrophoretic profiles of peptidase activities, rat intestinal brush-border membranes ( a ) and soluble cytoplasmic fraction (b). Details of solubilization of the enzyme and electrophoresis are described in the Methods section. The arrow shows the dye front.

were performed in the absence and presence of Ala (100 pmol/ml) and Gly-Pro (100 pmol/ml). The results showed that neither Ala nor Gly-Pro had an inhibitory effect on the rate of hydrolysis of any of the leucine-containing peptides (Table 6). Discussion

Earlier investigations with three tetrapeptides have indicated that tetrapeptides require brush-border membrane hydrolysis before transport by intestinal mucosal cells (Matthew & Adibi, 1976; Sleisenger et al., 1976; Adibi & Morse, 1977; Smithson & Gray, 1977). According to this hypothesis, we expected the tetrapeptide Leu-Gly-Gly-GIy to be hydrolysed by brush-border membrane aminopeptidases to free Leu and the tripeptide Gly-Gly-

Gly before intestinal uptake occurred. In the present study, a role for the brush-border membrane aminopeptidases in the mucosal hydrolysis of the tetrapeptide was confirmed, but, contrary to expectation, these experiments indicate that there also exists in rat small intestine a mucosal peptideuptake system capable of transporting significant amounts of the tetrapeptide in an intact form. It is now clear that mucosal uptake of peptides in the intact form and mucosal uptake of free amino acids are independent processes involving separate mechanisms (Silk, 1974; Matthews, 1975; Matthews & Adibi, 1976; Adibi, 1976). For the tetrapeptide Leu-Gly-Gly-Gly, however, we expected the liberated N-terminal Leu to be absorbed at the same rate as equivalent concentrations of Leu presented to the mucosa for absorption of the free amino acid. However, as shown in Table 1, at a concentration of 15 pmol/ml the rates of uptake of Leu from the test solution containing Leu-Gly-GlyGly and Leu-Gly were significantly greater than from the solutions of free Leu. Ala is a neutral amino acid known to share a common uptake system with Leu (Saunders & Isselbacher, 1966). At a concentration of 100 pmol/ml it was found to inhibit almost completely (94.5%) uptake of Leu from a test solution containing 10 pmol/rnl or less Leu (Table 1). At this concentration Ala did not affect brush-border peptidase activity against any of the three Leucontaining peptides (Table 6). In order to inhibit uptake of.free Leu released by the action of brushborder aminopeptidases on the Leu-containing peptides, Leu-Gly, Leu-Gly-Gly and Leu-Gly-GlyGly were perfused in the absence and presence of Ala at 100 pmol/ml. Ala caused an increase in appearance rate of free Leu during Leu-Gly and Leu-Gly-Gly perfusion, but the net uptake rates of Leu were unaffected by Ala (Table l), indicating that intact uptake was the major mode of transport of both Leu-Gly and Leu-Gly-Gly. Ala caused a substantial increase in the appearance of Leu

TABLE6 . Effect of alanine or Gly-Pro on peplidme activity of partially puriJied brush-borderfraction from rat jejunal mucosa Enzyme activity is expressed as ,urnol of leucine released mh-l mg-I of protein. Values are means & SEM from six experiments. Addition to reaction mixture

None

Alanine (100mmol/l) Gly-Pro (100 mmolh)

Enzyme activity against peptidases (unitshg of protein) Leu-Gly

Leu-Gly-Gly

Le~-Gly-Gly-Gly

0.52f 0.13 0.52f 0.17 0.54 ? 0.12

4.60 ? 0.75 4.98 f 0.87 5.03 f 0.66

0.90 f 0.10 0.84 f 0.11 0.89 k 0.09

Intestinal transport of a tetrapeptide

during Leu-Gly-Gly-Gly perfusion, indicating that significant quantities of Leu were released into solution during uptake of the tetrapeptide. However, total net uptake of Leu from the tetrapeptide at all concentrations examined was inhibited only about 50% by Ala at 100 pmol/ml. If sequential hydrolysis of the peptide from the Nterminus of the tetrapeptide by the luminal or brush-border membrane aminopeptidase were required for absorption of Leu, the N-terminal amino acid, its absorption would be inhibited more substantially by Ala Furthermore, if substantial carboxypeptidase or endopeptidase activities against this tetrapeptide were present in the intestinal lumen or brush-border membrane, other explanations for absorption of Leu from the tetrapeptide are possible. The absence of the expected products of these activities in the intestinal perfusate, however, precludes these possibilities. Thus these competition experiments indicate that the residual tetrapeptide-bound Leu is absorbed in an intact form by the rat small intestine unless there exists a transport system which will transport the Leu released by aminopeptidases in such a way that Ala cannot compete with Leu, or binding of the tetrapeptide to the brush-border membrane aminopeptidase alters the atlinity of Leu released by hydrolysis for the amino acid carrier such that Ala is less effective as an inhibitor. The absorption rate of Leu during perfusion of Leu-Gly-Gly-Gly (1 pmol/ml) in the presence of ANA (20 pmol/ml) was significantly (P < 0.005) lower than in its absence, indicating that brushborder aminopeptidase plays some role in the assimilation of Leu from the tetrapeptide. However, the extent of the inhibition of Leu absorption by ANA (24%) during the perfusion of Leu-Gly-GlyGly (1 pmol/ml) was relatively small. It is possible that at this concentration of ANA there still remains sufficient enzyme activity to hydrolyse tetrapeptide at 1-2 mmol/l. Because of the limited solubility of ANA at neutral pH, we could not examine this inhibition at higher concentration. Therefore it is possible that a higher concentration of ANA in the perfusion solution might have resulted in a more substantial inhibition of absorption. Since most amino nitrogen in the postprandial mesenteric and portal venous blood exists probably in the form of free amino acids, the Leu-Gly-GlyGly, when absorbed intact into the intestinal mucosal cells, must be hydrolysed to free amino acids. Therefore the soluble fraction of intestinal mucosal homogenates was examined for peptidase

9

activity with Leu-Gly-Gly-Gly as substrate. Substantial peptidase activity against the tetrapeptide 5). pHydroxywas detected (Table mercuribenzoate completely inhibits this enzyme activity, which makes it unlikely that the soluble fraction was contaminated with brush-border peptidases, which are not inhibited by this reagent (Heizer et al., 1972; Nicholson & Kim, 1975). Furthermore, peptidases in the soluble fraction had mobilities different from those of brush-border peptidases when the two fractions were subjected to polyacrylamide-gel electrophoresis (Fig. 3a and 3b). This is additional supportive evidence for the existence of peptidase activity against Leu-GlyGly-Gly in the cytoplasmic compartment of the rat intestinal mucosal cell. The polyacrylamide-gel electrophoresis data suggest that a cytoplasmic amino-oligopeptidase rather than a distinct tetrapeptidase is responsible for hydrolysing Leu-GlyGly-Gly, since enzyme activity for both Leu-Gly and Leu-Gly-Gly was present in both bands (Fig. 3b). The total hydrolytic capacity of the cytoplasmic peptidases (10-1 pmol of Leu-Gly-Gly-Gly hydrolysed min-I g-' of wet mucosa) determined in the assays in uitro (Table 5 ) far exceeded the rates of disappearance of the tetrapeptide during the perfusion experiment in uiuo (1.3 pmol min-' g-l of mucosa) (Table 1). Thus, if significant amounts of Leu-Gly-Gly-Gly are transported intact into the cell, there appears to be sficient cytoplasmic peptidase activity to complete hydrolysis of the peptide. As mentioned previously, intact uptake appeared to be the major mode of luminal disappearance of Leu-Gly and Leu-Gly-Gly under the conditions of our experiment. The question that arises is whether the tetrapeptide utilizes the same mucosal peptideuptake system or systems used for the intact transport of its related di- and tri-peptides. Gly-Pro, a dipeptide previously shown to be absorbed intact (Rubino, Field & Scwachman, 1971; Lane, Silk & Clark, 1975), did not inhibit brush-border peptidase activity against any of the substrates tested (Table 5), but had a similar and marked inhibitory effect on leucine uptake from both Leu-Gly and Leu-GlyGly. This is strong evidence that these three peptides share a common mucosal peptide-uptake system in the rat intestine. In contrast, Gly-Pro did not inhibit leucine uptake from Leu-Gly-Gly-Gly, which indicates that the mucosal peptide-uptake system for this tetrapeptide may be distinct from the system shared by its related di- and tri-peptide. For the di- and tri-peptides, there may be more than one transport mechanism in the intestine

10

Y. C. Chung, D. B. A . Silk and Y. S.Kim

(Matthews, 1975; Matthews & Adibi, 1976). For example, the absorption of carnosine, which was inhibited by several di- and tri-peptides, including Gly-Pro, was not affected by lysyl-lysine or glutamylglutamic acid (Addison, Burston & Matthews, 1973). Furthermore, uptake of glycine from Gly-Pro was not inhibited by Pro-Gly (Lane et al., 1975). These data suggest that there may be multiple di- and tri-peptide-transport systems in the small intestine. Whether there exists in mammalian intestine an additional peptide-uptake system uniquely available to tetrapeptides other than LeuGly-Gly-Gly cannot be determined from the data presented in this paper. The hypothesis that tetrapeptides undergo complete hydrolysis at the brush border before uptake of the constituent N-terminal amino acid and tripeptide was consistent with the previously reported absence of peptidase activity against tetrapeptides in the cytoplasmic fraction of rat intestinal mucosa (Kim et al., 1974; Schiller et al., 1977). Before the present study, the activity of cytoplasmic peptidases against only four (Ala-Gly-GlyGly, tetraglycine, Phe-Gly-Phe-Gly and Pro-PheGly-Gly) of the possible 160 000 tetrapeptides had been investigated (Kim et al., 1974). In light of the present results, which show the presence of cytoplasmic enzyme activity against the tetrapeptide, Leu-Gly-Gly-Gly, and the existence of a mucosal uptake system for the intact tetrapeptide, it is apparent that not all tetrapeptides are transported only as free amino acids and di- and tri-peptides after hydrolysis by brush-border aminopeptidases. However, only four tetrapeptides have been examined thus far and whether the tetrapeptide LeuGly-Gly-Gly possesses features common to many other tetrapeptides, and thus may serve as a model peptide in the investigation of general absorptive mechanisms, remains to be established.

Acknowledgments We are indebted to Dr James Whitehead and Dr Martha Anderson for critically reviewing the manuscript. This work was supported by Grant AM-17938 from the United States Public Health Service. References ADDISON,J.M., BURSTON,D. & MAITHEWS,D.M. (1973) Carnosine transport by hamster jejunum in vitro and its inhibition by other di- and tri-peptides. Clinical Science and Molecular Medicine, 4 5 , 3 4 .

ADIEI, S.A. (1971) Intestinal transport of dipeptides in man. Relative importance of hydrolysis and intact absorption. Journal of Clinical Investigation, 50,2266-2275. ADIEI, S.A. (1976) Intestinal phase of protein assimilation in man. American Journal of Clinical Nutrition, 29,205-2 15. ADrBi, S.A. & MERCER,D.M. (1973) Protein digestion in human intestine as reflected in luminal, mucosal and plasma amino acid concentration after meals. Journal of Clinical Investigation, 52,1586-1594. ADIEI, S.A. & MORSE,E.L. (1977) The number of glycine residues which limits intact absorption of glycine oligopeptides in human jejunum. Journal of Clinical Investigation. 60, 1008- 1016. BONLLIN, D.J., CRAMPTON, R.F., HEADING,E.E.& PELLING, D. (1973) Intestinal absorption of dipeptide containing glycine, phenylanine, proline, L-alanine or histidine in the rat. Clinical Science and Molecular Medicine, 45,845-858. CHEN, M.L., ROGERS,Q.R. & HARPER,A.E. (1962) Observation on protein digestion and absorption in vivo. IV. Further observations on the gastrointestinal contents of rats fed different dietary proteins. Journal of Nutrition, 76, 235241. CHENG, B., NAVAB, F., LIS, M.T., MULLER, T.N. & MAITHEWS, D.M. (1971) Mechanisms of dipeptide uptake by rat small intestine in vitro. Clinical Science, 40,247-257. DAHLQvIST, A. (1964) Method for assay of intestinal disaccharides. Analytical Biochemistry, 7, 18-25. DAVIS,B.J. (1964) Disc electrophoresis. 2. Method and application to human serum protein. Annals of the New York Academy of Sciences, 121,404-427. DONLON,J. & FORTRELL,P.F. (1972) Studies on substrate specificities and subcellular location of multiple forms of peptide hydrolases in guinea pig intestinal mucosa. Comparative Biochemistry and Physiology, B, 41,181-193. FUJITA,M., PARSONS, D.S. & WOJNAROWSKA, F. (1972) Oligopeptidases of brush border membranes of rat small intestinal mucosa cells. Journal of Physiology (London), 227,317-394. GRAY,G.M. & COOPER,H.L. (1971) Protein digestion and absorption. Gastroenterology, 61,535-544. HEIZER,W.D., KERLEY,R.L. & ISSELBACHER, K.J. (1972) Intestinal peptide hydrolase differences between brush border and cytoplasmic enzymes. Biochimica et Biophysica Acta, 2 6 4 , 4 5 0 4 6 I. JOSEFSSON, L. & SJOSTROM, H. (1966) Intestinal peptidases. IV. Studies on the release and subcellular distribution of intestinal dipeptidases of the mucosal cells of the pig. Acra Physiologica Scandinavica, 67,27-33. KANIA.R.K., SANTIAGO, N.A. & GRAY,G.M. (1977) Intestinal surface amino-oligopeptidases. 11. Substrate kinetic and topography of the active site. Journal of Biological Chemistry, 252,49294934. KIM, Y.S., BIRTWHISTLE, W. & KIM, Y.W. (1972) Peptide hydrolases in brush border and soluble fractions of small intestinal mucosa of rat and man. Journal of Clinical Investigation, 51, 1419-1430. KIM, Y.S.& BROPHY,E.J. (1976) Rat intestinal brush border peptidases. I. Solubilization, purification and physicochemical properties of two different forms of the enzyme. Journal of Biological Chemistry, 251,3 199-3205. KIM, Y.S.,KIM, Y.W. & SLEISENGER, M.H. (1974) Studies of the properties of peptide hydrolases in the brush border and soluble fractions of small intestinal mucosa in rat and man. Biochimica et Biophysica Acta, 370,283-296. KIM, Y.S.,MCCARTHY, D.M., LANE,W. & FONG,W.(1973) Alterations in the levels of peptide hydrolases and other enzymes in brush border and soluble fractions of rat small intestinal mucosa during starvation and refeeding. Biochimica el Biophysica Acta, 321,262-273. LANE.A.E., SILK,D.B.A. & CLARK,M.L. (1975) Absorption of two proline containing peptides by rat small intestine in vivo. Journal of Physiology (London), 248, 143-149. LANGLEY, R. ( 1968) Practical Statistics for Non-Mathematical People. Pan, London.

Intestinal transport of a tetrapeptide LOUVARD,D., MAROUX,S., RANNIER,CH. & DESNEULLE, P. (1975a) Topological studies on the hydrolases bound to the intestinal brush border membrane. 1. Solubilization by papain and Triton X-100. Biochimica et Biophysica Acta, 375,236248. LOWARD, D., MAROUX, S. & DESNUELLE,P. (1975b) Topological studies on the hydrolases bound to the intestinal brush border membrane. 2. Interactions of free and bound aminopeptidase with a specific antibody. Biochimica et Biophysica Acta, 389,389-400. LOWRY,O.H., ROSEBOROUGH, N.J., FARR,A.L. & RANDALL, R.J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193,265-275. M A ~ E W SD.M. , (1971) Protein absorption. Journal of Clinical Pathology, 24, (Suppl. 29-40). MAm?EWS, D.M. (1975) Intestinal absorption of peptides. Physiological Reviews, 55.537408. M A ~ E W SD.M. , & ADIBI, S.A. (1976) Peptide absorption. Gastroenterology, 71.15 1-16 1. J.A. & KIM, Y.S. (1975) A one step L-amino acid NICHOLSON, oxidase assay for intestinal peptide hydrolase activity. Analytical Biochemistry, 63,110-1 17. PETERS,T.J. (1970) The subcellular localization of di- and tripeptide hydrolase activity in guinea-pig small intestine. Biochemical Journal, 120,195-203. PETERS,T.J. (1973) The hydrolysis of glycine oligopeptides by guinea-pig intestinal mucosa and by isolated brush borders. Clinical Science and Molecular Medicine, 45,803-8 16. A. (1961) Significance of urinary PROCKOP,D.J. & SJOERDSMA, hydroxyprolie in man. Journal of Clinical Investigation, 40, 843-849. RUBINO,A., FIELD,M. & SCWACHMAN, H. (1971) Intestinal

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transport of amino acid residues of dipeptides. L. Influx of the glycine residue of glycyl-L-proline across mucosal border. Journal of Biological Chemistry, 246,3542-3548. ROBINSON,G.B. & SHAW. B. (1960) The hydrolysis of dipeptides by different regions of rat small intestine. Biochemical Journal, 77,35 1-356. K.J. (1966) Intestinal abSAUNDERS,S.J. & ISSELBACHER, sorption of amino acids. Gastroenterology, 50,586-595. SCHILLER, C.M., HUANG,T.1. & HEIZER,W.D. (1977) Isolation and characterization of four peptide hydrolases from the cytosol of rat intestinal mucosa. Gastroenterology, 72, 93100. SILK,D.B.A. (1974) Peptide absorption in man. Gut, 15,494501. SILK,D.B.A., PERREIT,D. & CLARK,M.L. (1974a) Intestinal transport of two dipeptides containing the same two neutral amino acids in man. Clinical Science and Molecular Medicine, 45,29 1-299. SILK,D.B.A., PERRETT,D.,WEBB,J.P. &CLARK,M.L.(1974b) Absorption of two tripeptides by the human small intestine: A study using a perfusion technique. Clinical Science and Molecular Medicine, 46,393-402. SLEISENGER, M.H., BURSTON,D., DALRYMPLE, A., WILKINSON, S. & MATTHEWS, D.M. (1976) Evidence for a single common carrier for uptake of a dipeptide and a tripeptide by hamster jejunum in vitro. Gastroenterology, 71,7682. SMITHSON, K.W. & GRAY,G.M. (1977) Intestinal assimilation of tetrapeptide in the rat. Journal of Clinical Investigation, 60,655-674. WINGATE,D.L., SANDBERG, R.J. & PHILLIPS,S.F. (1972) A comparison of stable and ITC-labelledpolyethylene glycol as volume indicators in the human jejunum. Gut, 13.812-815.

Intestinal transport of a tetrapeptide, L-leucylglycylglycylglycine, in rat small intestine in vivo.

Clinical Science (1979) 57, 1-1 1 Intestinal transport of a tetrapeptide, L-leucylglycylglycylglycine,in rat small intestine in vivo Y. C . C H U N...
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