Hospital, San Francisco, California 94121, and the Department of Medicine,

Transcription

1 J. Physiol. (1976), 258, pp With 1 plate and 1 text-figure Printed in Great Britain RELEASE OF PEPTIDE HYDROLASES DURING INCUBATION OF INTACT INTESTINAL SEGMENTS IN VITRO BY D. B. A. SILK AND Y. S. KIM From the Gastrointestinal Research Laboratory, Veterans Administration Hospital, San Francisco, California 94121, and the Department of Medicine, University of California, School of Medicine, San Francisco, California 94143, U.S.A. (Received 19 December 1975) SUMMARY 1. Rat intestinal segments have been incubated in isotonic saline in vitro and release of peptide hydrolase enzymes into the incubation media examined over a 90 min study period. 2. Chemical assay data, as well as analysis of electrophoretic mobilities of released enzymes on starch gel, indicate that peptide hydrolase enzymes in the incubation media originate predominantly from the cytoplasm of the mucosal cells. 3. Peptide hydrolases were released rapidly from intact intestinal segments. Release occurred from the start of the in vitro incubations and was not affected by temperature and shaking. 4. It is concluded that compared to the in vivo situation, cytoplasmic peptide hydrolases are released from intestinal tissue very rapidly in vitro. Caution is therefore required when comparing results of in vivo and in vitro peptide absorption experiments. INTRODUCTION Experimental evidence indicates that the brush border as well as the cytosol fraction of intestinal mucosa contains enzymes capable of hydrolysing small peptides and both groups of enzymes have been suggested to play a role in the terminal stages of protein digestion (Rhodes, Eichholz & Crane, 1967; Peters, 1970, 1973; Donlon & Fottrell, 1972; Kim, Birtwhistle & Kim, 1972; Fujita, Parsons & Wojnarowska, 1972; Wojnarowska & Gray, 1975). The luminal contents of animal and human small intestine also contain peptide hydrolases (Josefsson, Lindberg & Ojesj6, 1968; Heizer & Laster, 1969; Silk & Kim, 1975; D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). The jejunum is the major site of protein absorption in vivo (Borgstr6m,

2 490 D. B. A. SILK AND Y. S. KIM Dahlqvist, Lundh & Sj6vall, 1957; Nixon & Mawer, 1970). In this region of the intestine in vivo luminal enzymes originate predominantly from cytoplasm of intestinal mucosal cells (Silk & Kim, 1975; D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). However, the enzyme activity is low and does not significantly contribute toward the hydrolysis of peptides present in the gut lumen (D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). In contrast to the in vivo situation, reports indicate that in vitro, mucosal peptide hydrolases are released rapidly into medium bathing intestinal mucosa (Josefsson & Sj6str6m, 1966; Lindberg, Noren & Sj6str6m, 1975). It is not known, however, whether these enzymes originate from the brush border or the cytoplasm of the mucosal cell. The present study was therefore carried out to determine the origin of mucosal peptide hydrolases released from intact intestinal in vitro, and to assess the magnitude of this enzyme release. The results show that peptide hydrolases released from intact mucosa in vitro originate predominantly from the cytoplasm of mucosal cells, and only a small amount of the enzyme activity appears to be derived from intestinal brush borders. The magnitude of enzyme release in vitro is considerable as compared to that in vivo, indicating that serious consideration should be given to this property of mucosal peptide hydrolases when interpreting results of in vitro dipeptide absorption studies. METHODS Unfasted female Wistar rats ( g) were used throughout. Immediately after sacrifice, whole small intestine was removed, and divided equally into two segments; segments were washed with 50 ml. isotonic saline (40 C) and slit longitudinally. In the first series of experiments, intestinal segments were placed for six successive 15.min periods in 20 ml. aliquots of isotonic saline maintained at 0-4 C in a cold room. In this way enzyme release was studied for a total 90-min time period. Cytosol peptide hydrolase enzymes are unstable (Kim et al. 1972). Assays of enzyme activities in the media were therefore performed immediately after removal of intestinal segments. At the end of the incubation experiments, intestinal segments were scraped on a chilled glass plate, the mucosal scrapings weighed and immediately homogenized in isotonic saline (10 mk/g) as previously described (Kim et al. 1972; D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). After homogenization, samples were then sonicated for 15 sec at 50 W with a probe terminal diameter of 3 mmn (Branson Sonic Power Co., Plainview, N.Y., U.S.A.). Suitably, diluted aliquots were then assayed for peptide hydrolase activity. In the second series of experiments, intestinal segments were placed in aliquots of isotonic saline maintained at 37 C in a shaking water-bath (50 oscillations/min). Experiments were then carried out exactly as described above. For the zymogram studies (see below) intestinal segments were placed in 20 ml. isotonic saline (4 and 370 C) for 2 hr. Aliquots of media were stored at -20 C until required for starch gel electrophoresis. Choice of 8ub8trate8 and am8ay procedures. The dipeptides glycyl-l-phenylalanine (Gly-Phe) and L-phenylalanyl-glycine (Phe-Gly) were chosen as substrates for

3 PEPTIDE H YDROLASE RELEASE determination of peptide hydrolase activity because mucosal brush border peptide hydrolase activity can be readily distinguished from mucosal cytosol activity by the differential sensitivities of the enzymes in these two fractions to the inhibitory effect of p-hydroxymercuribenzoate (PHMB; Heizer, Kerley & Isselbacher, 1972). Aliquots of saline media and mucosal homogenates were assayed for peptide hydrolase activity in the absence and presence of 0 5 mm-phmb by the method of Nicholson & Kim (1975) using 6*25 mu substrate concentrations. Linear kinetics were obtained at these substrate concentrations. Brush border peptide hydrolase activity is that activity observed in the presence of 0.5 mm-phmb. Cytosol peptide hydrolase activity is the activity in the absence of PHMB minus the activity in the presence of the inhibitor (D.B.A. Silk, J. A. Nicholson and Y. S. Kim, submitted). Aliquots of saline media and mucosal homogenates were also assayed for sucrase activity. The method of Dahlqvist (1968) was used. Zymogram method. Vertical starch gel electrophoresis was performed with Buchler gel electrophoresis apparatus (Buchler Instruments, Fort Lee, N. J., U.S.A.) by the method of Lewis & Harris (1967) at ph 7*4 (bridge buffer 0-1 M Tris-maleate; gel buffer M Tris-maleate). Aliquots, 0 04 ml, of the following solutions were applied to the gels: (1) saline incubation media; (2) solutions containing intestinal mucosal cytosol; and (3) solubilized intestinal brush border peptide hydrolase enzymes. Solutions (2) and (3) were prepared as previously described (D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). After application of enzymes, eleotrophoresis was performed in a cold room at 40 C for 20 hr at a potential of 5 V/cm. The starch gel was sliced into two layers after electrophoresis. The reaction mixture containing the dipeptide under examination (Gly-Phe, Phe-Gly or L-leucyl-L-leucine (Leu-Leu), L-amino acid oxidase, horseradish peroxidase and O-dianisidine) was mixed with an equal volume of 2 % aqueous agar (550 C) and poured over the sliced surfaces of the gel. The starch gel with its agar overlay was then incubated for 1-2 hr at 350 C. Calculaion of data. The amounts of peptide hydrolase enzymes released from intestinal segments are expressed in terms of jamole dipeptide substrate (Gly-Phe, Phe-Gly) hydrolysed in 10 min by enzymes in the total volume of media exposed to intestine. In each individual experiment, intestinal segments were incubated for a total of six successive 15.min periods in separate aliquots of saline. Thus enzyme released at 60 min, for example, is the sum of enzyme activity released into the first four aliquots of medium. RESULTS Peptide hydrolase activities in saline media Text-fig. 1 shows the total activity of peptide hydrolase enzymes (#mole dipeptide hydrolysed per 10 min) released from intestinal segments as a function of time. Release of Gly-Phe and Phe-Gly hydrolase activity into saline media maintained at 0-4 and 370 C was linear with respect to time over the 90 min study period. The media contained much greater amounts of Gly-Phe hydrolase activity than Phe-Gly hydrolase activity. Similar proportions of intestinal mucosal Gly-Phe ( s.e. of mean 2-6 %) and Phe-Gly ( %) hydrolase activities were released into saline media maintained at 0-4 C by the end of the 90 min study period. In contrast a significantly higher proportion of intestinal mucosal Gly-Phe ( %) i8 I8 P HY

4 492 D. B. A. SILK AND Y. S. KIM than Phe-Gly ( %) hydrolase activity was released into saline media maintained at 370 C (t = 2-81, P < 0 05; paired t test). Table 1 shows the proportions of intestinal mucosal peptide hydrolase activity released that was sensitive and resistant to the intestinal mucosal cytosol peptide hydrolase inhibitor, PHMB. The bulk of enzyme activity ( > 83-9 %) released into saline media maintained at 0-4 and 370 C was sensitive to PHMB. 400i C E C> o -c * a 200 E C4 E V W c N 40 C Time (min) C Text-fig. 1. Amounts of peptide hydrolase released from intestinal segments incubated in isotonic saline. Filled circles refer to jcmole Phe-Gly hydrolysed in 10 min by the total amount of enzymes released into incubation media (maintained at 4 or 37 C). Open circles refer to molee Gly-Phe hydrolysed in 10 min by total amounts of enzyme released into the same incubation media. Vertical bars represent 1 s.m of the mean values of five individual studies. Zymogram 8tudie8 It has previously been shown, using the same zymogram technique as we have employed during the present study, that rat intestinal mucosal cytosol and brush border peptide hydrolase enzymes have different electrophoretic mobilities (Kim et al. 1972; D. B. A. Silk, J. A. Nicholson

5 PEPTIDE HYDROLASE RELEASE 493 and Y. S. Kim, submitted). Peptide hydrolases released from intact intestine into saline media during the present study consistently had the same electrophoretic mobilities as intestinal cytosol, and not intestinal brush border peptide hydrolase enzymes. This was the case with all three substrates (Gly-Phe, Phe-Gly and Leu-Leu) used in this study. PI. 1 shows a representative zymogram using Leu-Leu as substrate. The electrophoretic mobilities of enzymes released into the medium (Slot 1) are TABiE 1. Proportions of peptide hydrolase activity detected in the saline media that were sensitive to P11MB and resistant to P11MB. Individual values (mean, n = 5) are % peptide hydrolase activity sensitive to and resistant to P11PM Temperature of media ' C 370 C A~~~~~ 5 Substrate P11MB sensitive PHMB resistant P1MB sensitive P11MB resistant time,,,- A, r (min) Gly-Phe Phe-Gly Gly-Phe Phe-Gly Gly-Phe Phe-Gly Gly-Phe Phe-Gly * * * '3 9* * distinct to papain solubilized brush border peptide hydrolases obtained from two different animals (Slots 2, 3). Significant staining was noted at the o.-igin (Slot 1) where aliquots of saline media were placed. Recent experience in this laboratory indicates that this is due to small amounts of membrane-bound brush border peptide hydrolases. The results of the zymogram studies taken in conjunction with the chemical assay data indicate that peptide hydrolases released from intact intestinal tissue in vitro predominantly originate from the cytoplasm of mucosal cells rather than the brush border. Release of bru8h border peptide hydrolase8 and 8ucrase It is evident from Table 1 that although the bulk of peptide hydrolase enzyme activity released from intestinal tissue originates from the cytoplasm of mucosal cells, the saline media always contained some brush border peptide hydrolase activity. Table 2 shows the proportion of total intestinal mucosal brush border peptide hydrolase activity released into the saline media during the study period. Less than 5 % Phe-Gly brush border peptide hydrolase activity was released and 10% or less of Gly-Phe brush border peptide hydrolase activity. To confirm that brush border enzymes remain tightly bound to the microvillous membrane during incubation of intestinal segments in vitro, sucrase activities in the saline i8-2

6 494 D. B. A. SILK AND Y. S. KIM media and intestinal mucosa were also compared. During the 90 min incubation period, 1 1 % or less of total intestinal mucosal sucrase activity was released. TABLE 2. Proportion of intestinal mucosal brush border enzymes released into saline media. Individual values (mean ± 1 S.E. of mean, n = 5) are % mucosal brush border enzyme activity released into incubation media Temperature of media C 370 C Peptide hydrolase Peptide hydrolase Enzyme... Sucrase Gly-Phe Phe-Gly Sucrase Gly-Phe Phe-Gly Time of incubation (min) ±3 0-2± ± ± ± ± ± ±01 5-5± ± ±2 5-8± ± ± ± ± ± ± ± ± ± ± ± ± ±0-7 DISCUSSION Josefsson & Sj6str6m (1966) demonstrated that intestinal mucosal dipeptidase activity was released rapidly from intact pig small intestine into 0-25 M sucrase solution maintained at 40 C. They suggested that the enzymes in the incubation media could originate from the brush border of the mucosal cell. The results of the present study do not support their conclusion. Heizer et al. (1972) showed, using Gly-Phe and Phe-Gly as substrates, that PHMB abolishes intestinal mucosal cytosol peptide hydrolase activity whereas brush border peptide hydrolase activity is unaffected. These findings have subsequentlybeen confirmed in this laboratory (Nicholson & Kim, 1975). Data presented in Table 1, showing greater than 96 % inhibition of Gly-Phe peptide hydrolase activity in saline media by PHMB, and greater than 84% inhibition of Phe-Gly peptide hydrolase activity, suggest that the released peptide hydrolase activity in saline media originates predominantly from the cytoplasm of the mucosal cells. Solubilized rat brush border and cytoplasmic peptide hydrolases have different electrophoretic mobilities on starch gel when the method of Lewis & Harris is used (Kim et al. 1972; D. B. A. Silk, J. A. Nicholson and Y. S. Kim, submitted). Throughout the present study, enzymes released into saline media had the same electrophoretic mobilities as rat intestinal cytosol peptide hydrolases. Thus, both the assay and zymogram data indicate the peptide hydrolase enzyme released from intact rat intestinal tissue in vitro predominantly arises from the cytoplasmic compartment of

7 PEPTIDE HYDROLASE RELEASE 495 mucosal cells. It is of interest in the light of those conclusions that Clark & Porteous (1965) showed that two other cytosol enzymes (lactate dehydrogenase, and phosphoglucoisomerase) were also rapidly released during the in vitro isolation of intestinal mucosal cells. The assay data (Tables 1, 2) support the release of small quantities of brush border peptide hydrolases during in vitro incubation of intestinal segments. It is unlikely that these enzymes exist in the solubilized form however; significant staining at the origin of the starch gels (P1. 1, Slot 1) indicates the presence of membrane-bound brush border peptide hydrolases (Kim et al. 1972). There is a suggestion that greater proportions of brush border peptide hydrolases than sucrase were released (Table 2). Absolute amounts of enzymes released were too low however to draw conclusions about the possibility of differential binding of surface enzymes to the microvillous membrane. The lack of significant quantities of brush border enzymes in the saline media is in keeping with the contention that both disaccharidases and dipeptide hydrolases are tightly bound to the microvillous membrane of the mucosal cell (Forstner, Sabesin & Isselbacher, 1968; Kim et al. 1972; Louvard, Maroux, Vannier & Desnuelle, 1975). It is evident from the results that cytoplasmic peptide hydrolases are released from intestinal segments incubated in vitro in considerable quantities (100 smole Gly-Phe hydrolysed in 10 min by enzymes released 30 min after the start of the incubations), and the release did not appear to be affected by temperature (Text-fig. 1) or shaking. During the last few years, much attention has been focused on the absorption of peptides as a major mode of absorption of protein digestion products (Adibi, 1971; Matthews, 1971, 1975; Silk, 1974), and the results of many in vivo and in vitro experiments in the field of peptide absorption have now been reported. In the in vivo situation, peptide hydrolases appear to be released slowly from intact intestinal tissue (Adibi, 1971; Silk, Perrett & Clark, 1973; Lindberg et al. 1975). Thus, only minor components of peptides under examination are hydrolysed by 'luminal' enzymes. In contrast, release of mucosal peptide hydrolase enzymes of the magnitude demonstrated in this and other studies (Josefsson & Sjostrom, 1966; Lindberg et al. 1975) may be sufficient to hydrolyze major components of peptide applied to the mucosal medium during in vitro experiments. Thus, in view of the marked differences between in vivo and in vitro rates of release of mucosal cytosol peptide hydrolase enzymes, caution is required when comparing results of in vivo and in vitro peptide absorption experiments.

8 496 D. B. A. SILK AND Y. S. KIM The technical assistance of Miss Eleanor Judelson is gratefully acknowledged. D. B. A. Silk is in receipt of a Medical Research Council of Great Britain Travelling Fellowship. This work was supported by Public Health Service Grant AM from the National Institute of Health. REFERENCES ADmBI, S. A. (197 1). Intestinal transport of dipeptides in man: relative importance of hydrolysis and intact absorption. J. clin. Invest. 50, BORGSTROM, B., DATTQVIST, A., LUNDH, G. & SJWvALL, J. (1957). Studies on intestinal digestion and absorption in the human. J. cdin. Inve8t. 36, CLARM, B. & POBTEOUS, J. W. (1965). The isolation and properties of epithelial cell 'ghosts' from rat small intestine. Biochem. J. 96, DAHLQVIST, A. (1968). Assay of intestinal disacharidases. Analyt. Biochem. 22, DO wlon, J. & FoTnRELL, P. F. (1972). Studies on substrate specificities and subcellular location of multiple forms of peptide hydrolases in guinea-pig intestinal mucosa. Comp. Biochem. Physiol. 41B, FoRsTEn, G. G., SABESIN, S. M. & ISsELBACHER, K. J. (1968). Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem. J. 106, FuJITA, M., PAsoNs, D. S. & WoJNARowsKA, F. (1972). Oligopeptidases of brush border membranes of rat small intestinal mucosal cells. J. Physiol. 227, HEJZER, W. D. KmaLEY, R. L. & ISSELBACHER, K. J. (1972). Intestinal peptide hydrolases. Differences between brush border and cytoplasmic enzymes. Biochim. biophys. Ada 264, HzEIzR, W. D. & LASTER, L. (1969). Peptide hydrolase activities of the mucosa of human small intestine. J. din. Invest. 48, JoSEFssoN, L., LINDBERG, T. & O)JEsJ6, L. (1968). Intestinal dipeptidase. Dipeptidase activities in human juice. Scand. Jnl Gastroenterol. 3, JOsElrssN, L. & SJOSTROM, H. (1966). Intestinal dipeptidases. IV. Studies on the release and subcellular distribution of intestinal dipeptidases of the mucosa cells of the pig. Ada physiol. 8cand. 67, Kim, Y. S., BLRTWHISTLE, W. & KEM, Y. W. (1972). Peptide hydrolases in the brush border and soluble fractions of small intestinal mucosa of rat and man. J. dlin. Invest. 51, LEwIs, W. H. P. & HARRis, H. (1967). Human red cell peptidases. Nature, Lond. 215, LINDBERG, T., NoiRN, 0. & SJOSTROM, H. (1975). Peptidases of the intestinal mucosa. In Peptide Transport in Protein Nutrition, ed. MArrTEws, D. M. & PAYNE, J. W., pp Amsterdam, New York: North Holland-American Elsevier. LOUVARD, D., MARoux, S., VEEwmR, CH. & DESNUJTx, P. (1975). Topological studies of the hydrolases bound to the intestinal brush border membrane. 1. Solubilization by papain and Triton X-100. Biochim. biophys. Ada 375, MATmEw, D. M. (1971). Protein absorption. J. clin. Path. 5, suppl. 24, 5, MATTHEws, D. M. (1975). Absorption of peptides by mammalian intestine. In Peptide Transport in Protein Nutrition, ed. MAsrriEws, D. M. & PAYNE, J. W., pp Amsterdam, New York: North Holland-American Elsevier. NICHOLSON, J. A. & KIM, Y. S. (1975) A one step L-amino acid oxidase assay for intestinal peptide hydrolase activity. Analyt. Biochem. 63,

9 r q *,v _. - X Se, - s!. -ok i,~~~~~~~~~~~~~~~~~~ The Journal of Physiology, Vol. 258, No. 2 Plate 1 Milan.: f:.i.-. ; D. B. A. SILK AND Y. S. KIM (Facing p. 497)

10 PEPTIDE HYDROLASE RELEASE 497 NIXON, S. E. & MAwER, G. E. (1970). The digestion and absorption ofprotein in man. 1. The site of absorption. Br. J. Nutr. 24, PLEBw, T. J. (1970). The subeellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine. Bioclem. J. 120, PETERs, T. J. (1973). The hydrolysis of glycine oligopeptides by guinea-pig intestinal mucosa and by isolated brush borders. CJin Sci. 45, RHODEMS, J. B., EICHmoLz, A. & CRANE, R. K. (1967). Studies on the organization of the brush border in intestinal epithelial cells. IV. Amino-peptidase activity in microvillous membranes of hamster intestinal brush border. Biochim. biophya. Acta 135, Sno, D. B. A. (1974). Peptide absorption in man. Gut 15, Sux, D. B. A. & Kim, Y. S. (1975). A study of intraluminal peptide hydrolase activity in the rat. Olin. Sci. 49, Sux, D. B. A., Pxewr, D. & CLAse, M. L. (1973). Intestinal transport of two dipeptides containing the same two neutral amino acids in man. Clin. Sci. 45, WojwNowsiu, F. & GRAY, G. M. (1975). Intestinal surface peptide hydrolases. Identification and characterization of three enzymes from rat brush border. Biochim. biophy8. Acta 403, EXPLANATION OF PLATE Zymograms of peptide hydrolase enzymes released from intact intestinal segments in vitro (Slot 1) and solubilized intestinal mucosal brush border peptide hydrolases (Slots 2, 3) prepared from two different animals. Leu-Leu used as substrate.