Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.     

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes

Summary In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.     

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.     

Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.     

Possible precursors of aminopeptidase and alkaline phosphatase in the proximal tubules of kidney and the crypts of small intestine of mice

Summary Two hydrolytically inactive proteins, one having common antigenic determinants with aminopeptidase and the other with alkaline phosphatase, have been localised in the apical cytoplasm of crypt cells of small intestine and in the cytoplasm of proximal convoluted tubules. In addition, the two proteins are also differently heat labil. Although they could not be detected with mere histochemical stain methods, they were detected by the immunofluorescence sandwich technique using specific antibody directed against either of the solubilised enzymes. The findings were confirmed using the previously described immunohistochemical method (Wachsmuth, 1973). The cellular and subcellular localisation implies that these two proteins are precursors of the hydrolytically active brush border membrane enzymes.     

Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking

Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical membrane trafficking in enterocytes. Cultured mucosal explants of pig small intestine were treated for 2 h with the cholesterol sequestering agent methyl-beta-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. The treatment reduced the cholesterol content >50%. Morphologically, the Golgi complex/trans-Golgi network was partially transformed into numerous 100-200 nm vesicles. By immunogold electron microscopy, aminopeptidase N was localized in these Golgi-derived vesicles as well as at the basolateral cell surface, indicating a partial missorting. Biochemically, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking.     

Separation and characterization of a dipeptidyl aminopeptidase that degrades enkephalins from monkey brain

A dipeptidyl aminopeptidase (a membrane-bound enzyme) which cleaved Met-enkephalin and released dipeptide (Tyr-Gly) was partially purified from monkey brain. A fraction containing both exoaminopeptidase and dipeptidyl aminopeptidase activity was obtained from DE-52 cellulose column chromatography. The dipeptidyl aminopeptidase activity in this fraction was not inhibited by addition of bestatin (300 μg/ml), while the exoaminopeptidase was strongly inhibited. Both enzymes were separated by AH-Sepharose 4B column chromatography. The molecular weight of the dipeptidyl aminopeptidase was calculated about 110,000. The enzyme activity was inhibited by addition of diisopropylfluorophosphate (DFP) or o-phenanthroline.     

Brush border membrane hydrolysis of S-benzyl-cysteine-p-nitroanilide, and activity of aminopeptidase M

A peptidase activity which can hydrolyze cysteinylglycine and S-benzyl-cysteine-p-nitroanilide was purified from rat renal brush border membranes. The purified peptidase exhibits an even greater specific activity when assayed with substrates for aminopeptidase M, leucine-p-nitroanilide and alanine-p-nitroanilide. All three activities copurify and coelectrophorese. In addition titration of the three activities in isolated brush border membrane vesicles with Fab antibodies prepared against the highly purified peptidase produced similar inactivation profiles. Therefore, the activity within brush border membrane towards S-benzyl-cysteine-p-nitroanilide is due to the action of aminopeptidase M (EC 3.4.11.2.)     

Interaction of purified brush-border membrane aminopeptidase N and dipeptidyl peptidase IV with lectin-sepharose derivatives

The glycoprotein nature of two peptidases purified from the rat intestinal brush-border membrane was examined by their interaction with several lectin-Sepharose derivatives. Aminopeptidase N (EC 3.4.11.2), which contains 20% carbohydrate by weight, was bound minimally (less than 30%) by columns of Con A-, RCAI- and WGA-Sepharose. Alternatively, a greater proportion of dipeptidyl peptidase IV (EC 3.4.14.-) was bound by these immobilized lectins with 50% of the enzyme binding to Con A-Sepharose. Treatment of both enzymes with neuraminidase enhanced the binding of aminopeptidase to RCAI-Sepharose by 4-fold but did not alter the binding patterns of dipeptidyl peptidase IV. A sequential fractionation of the two peptidases with columns of Con A- and RCAI-Sepharose gave four fractions of each enzyme with differing lectin-binding specificities. Approximately 60% of the dipeptidyl peptidase IV interacted with either one or both of the lectins while only 30% of the aminopeptidase N did so. Kinetic analysis of the four isolated fractions revealed some differences, possibly related to variations in the carbohydrate moiety. The findings confirm that these two purified rat intestinal brush-border membrane peptidases are glycoproteins and, while they share a common physiologic function and source, they apparently have very different and possibly unique asparagine-linked oligosaccharide side-chains. In addition, a considerable degree of microheterogeneity exists in the carbohydrate structure of these two enzymes.     

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar membrane and a weak intracellular staining was demonstrated. No staining was detected in the basolateral membrane. Likewise, the immunogold labelling on Epon-embedded sections was concentrated in the microvillar membrane, whereas the basolateral membrane did not contain significant amounts of labelling. Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar membrane by smooth vesicles having a diameter about 70 to 100 nm and does not pass the basolateral membrane on its way to the brush border membrane.     

Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells.

To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15--20% and a 10-fold enrichment of the plasma membrane markers 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase chosen to follow their purification. The four fractions were constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes, led to the conclusion that the (Na+ + K+)-dependent ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5'-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, dissacharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane.     

Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.     

Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human cœliac disease

Cœliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.     

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.     

Radiation inactivation analysis of kidney microvillar peptidases

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, gamma-glutamyl transferase, presented a target size corresponding to the dimeric Mr. The other enzymes are known to be homodimers. Three of these, aminopeptidase A. aminopeptidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopeptidase-24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.     

Prednisolone enhances aminopeptidase turnover in adult rat small intestine

In adult male rats, fed prednisolone (0.75 mg/kg/day) for 7 days, brush border aminopeptidase activity was increased (P < 0.001) by 106% compared to pair-fed controls. [¹⁴C]Tyrosine was injected intraperitoneally 16 h and [³H]tyrosine 6 h before death. The ³H/¹⁴C ratio was 1.79 ± 0.21 (S.D.) in purified microvillus membranes from treated rats compared to 1.30 ± 0.16 (P < 0.01) in controls. Polyacrylamide gel electrophoresis of brush border membranes under denaturing conditions showed that the increased double-isotope ratio in membranes from treated rats was mainly in the high molecular weight protein subunits (> 80 kDa) Detergent-solubilized aminopeptidase was purified after in vivo labeling by protein A-Sepharose-antiaminopeptidase affinity chromatography. The ³H/¹⁴C ratio in aminopeptidase was 2.42 ± 0.15 (P < 0.05) in treated rats compared to 1.63 ± 0.13 in controls. Over the experimental period steady-state isotope reutilization and protein labeling was demonstrated and there was no isotope metabolism. Total microvillus membrane lipid content was unaffected by prednisolone. We conclude that prednisolone increases brush border aminopeptidase activity by increasing enzyme turnover. Other high molecular weight brush border proteins were similarly affected.