Frank Girbig

Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and β-lactam antibiotics

The interaction of two renin inhibitors, S 86,2033 and S 86,3390, with the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86,3390 and 2.5 mM of S 86,2033 led to a half-maximal inhibition of the H(+)-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86,2033 and S 86,3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of S 86,3390 was stimulated by an inwardly directed H(+)-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86,2033 and 86,3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent Mr 127,000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and beta-lactam antibiotics.

Differential interaction of glimepiride and glibenclamide with the β-cell sulfonylurea receptor II. Photoaffinity labeling of a 65 kDa protein by [3H]glimepiride

Glimepiride is a novel sulfonylurea for the treatment of type II-diabetic patients exhibiting different receptor binding kinetics to beta-cell membranes with 8-9-fold higher koff rate and 2.5-3-fold higher kon rate compared to glibenclamide (see accompanying paper (Müller, G. et al. (1994) Biochim. Biophys. Acta 1191, 267-277)). To elucidate the molecular basis for this differential behaviour of glimepiride and glibenclamide, direct photoaffinity labeling studies using beta-cell tumor membranes were performed. [3H]Glimepiride was specifically incorporated into a membrane polypeptide of M(r) = 65,000 under conditions, which led to predominant labeling of a 140 kDa protein by [3H]glibenclamide (Kramer, W. et al. (1988) FEBS Lett. 229, 355-359). Labeling of the 140 kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65 kDa protein by [3H]glimepiride. The 65 kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33 kDa protein. Competitive Scatchard analysis of [3H]glimepiride-binding and [3H]glibenclamide-binding to RINm5F cell membranes using glibenclamide and glimepiride, respectively, as heterologous displacing compounds yielded non-linear plots. These findings may be explained by cooperative interactions between the 140 and 65 kDa sulfonylurea-binding proteins. The possibility that sulfonylureas of different structure have different access to the 140 and 65 kDa receptor proteins due to the beta-cell membrane barrier was investigated by photoaffinity labeling of solubilized beta-cell membrane proteins. Interestingly, solubilization of beta-cell tumor membranes led to a shift of specific [3H]glibenclamide binding from the 140 kDa to the 65 kDa binding protein, exclusively, and to an increased labeling of the 65 kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd values measured for both sulfonylureas upon solubilization of beta-cell tumor and RINm5F cell membranes (see accompanying paper). Furthermore, competitive Scatchard plots of [3H]glimepiride binding to solubilized RINm5F cell membrane proteins in the presence of glibenclamide and vice versa approximate linearity suggesting loss of cooperativity between the 140 kDa glibenclamide-binding and 65 kDa glimepiride-binding proteins upon solubilization. The physiological significance of the differential interaction of glimepiride and glibenclamide with different binding proteins was also substantiated by photoaffinity labeling of RINm5F cells leading to labeling of a 140 kDa protein by [3H]glibenclamide and of a 65 kDa protein by [3H]glimepiride.(ABSTRACT TRUNCATED AT 400 WORDS)

Intestinal absorption of β‐lactam antibiotics and oligopeptides

The H(+)-dependent uptake system responsible for the enteral absorption of oligopeptides and orally active beta-lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n-octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active alpha-amino-cephalosporin, D-cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein-dependent. In these proteoliposomes the binding protein for oligopeptides and beta-lactam antibiotics of Mr 127,000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles. The uptake system for beta-lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D-cephalexin was taken up by intact brush border membrane vesicles, whereas the L-enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127-kDa binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127-kDa protein in the presence of both enantiomers. After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127-kDa binding protein showed a significant uptake of D-cephalexin whereas the L-enantiomer was not transported. The uptake rates for D-cephalexin into proteoliposomes correlated with the content of 127-kDa binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127-kDa binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored. These results indicate that the binding protein for oligopeptides and beta-lactam antibiotics of Mr 127,000 mediates the stereospecific and H(+)-dependent transport of orally active beta-lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127-kDa binding protein is the intestinal peptide transport system (or a component thereof).

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2‐azetidinone cholesterol absorption inhibitors. An integral membrane protein of M r 145.3±7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration‐dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D‐glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2‐azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M r 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.

Characterization and chemical modification of the Na+-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.

Intestinal uptake of dipeptides and β-lactam antibiotics. I, The intestinal uptake system for dipeptides and β-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.

Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure.

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His100-Thr101-Ser102using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112–128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.

Direct photoaffinity labelling of binding proteins for β-lactam antibiotics in rabbit intestinal brush border membranes with [3H]benzylpenicillin

Brush border membrane vesicles from rabbit small intestine were used to study the intestinal uptake system for beta-lactam antibiotics. Benzylpenicillin inhibited the H+-dependent uptake of alpha-aminocephalosporins in a concentration-dependent manner suggesting a common transport system for alpha-aminocephalosporins and benzylpenicillin. Benzylpenicillin is therefore a suitable probe to characterize this transport system. Irradiation of [3H]benzylpenicillin using light sources having their maximum of radiation at 300 or 254 nm resulted in a covalent incorporation of radioactivity into penicillin binding proteins as was shown with serum albumin. Hence [3H]benzylpenicillin can be used for direct photoaffinity labeling of penicillin binding proteins in different cells and tissues. In brush border membrane vesicles from rabbit small intestine predominantly a membrane polypeptide with an apparent molecular weight of 127,000 was labeled by [3H]benzylpenicillin. Competition labeling experiments demonstrated that beta-lactam antibiotics--penicillins and cephalosporins--specifically interact with this protein, whereas amino acids, sugars or bile acids had no effect on the labeling pattern. Compounds which decreased the labeling of the 127,000 molecular weight membrane polypeptide also inhibited the H+-dependent uptake of the alpha-aminocephalosporin cephalexin into intestinal brush border membrane vesicles. These results suggest that a polypeptide of molecular weight 127,000 in the brush border membrane from rabbit small intestine is a constituent of a common transport system responsible for the uptake of orally effective beta-lactam antibiotics and dipeptides. beta-Lactam antibiotics which are not absorbed from the small intestine also bind from the luminal site to this transport system, but are not transported across the brush border membrane.

Intestinal cholesterol absorption: identification of different binding proteins for cholesterol and cholesterol absorption inhibitors in the enterocyte brush border membrane.

Absorption of cholesterol from the intestine is a central part of body cholesterol homeostasis. The molecular mechanisms of intestinal cholesterol absorption and the proteins mediating membrane transport are not known. We therefore aimed to identify the proteins involved in intestinal cholesterol absorption across the luminal brush border membrane of small intestinal enterocytes. By photoaffinity labeling using photoreactive derivatives of cholesterol and 2-azetidinone cholesterol absorption inhibitors, an 80-kDa and a 145-kDa integral membrane protein were identified as specific binding proteins for cholesterol and cholesterol absorption inhibitors, respectively, in the brush border membrane of small intestinal enterocytes. The 80-kDa cholesterol-binding protein did not interact with cholesterol absorption inhibitors and vice versa; cholesterol or plant sterols did not interfere with the 145-kDa molecular target for cholesterol absorption inhibitors. Both proteins showed an identical tissue distribution and were exclusively found at the anatomical sites of cholesterol absorption-duodenum, jejunum and ileum. Neither stomach, cecum, colon, rectum, kidney, liver nor fat tissue expressed the 80- or 145-kDa binding proteins for cholesterol and cholesterol absorption inhibitors. Both proteins are different from the hitherto described candidate proteins for the intestinal cholesterol transporter,-SR-BI, ABC G5/ABC G8 or ABC A1. Our data strongly suggest that intestinal cholesterol absorption is not facilitated by a single transporter protein but occurs by a complex machinery. Two specific binding proteins for cholesterol (80 kDa) and cholesterol absorption inhibitors (145 kDa) of the enterocyte brush border membrane are probable protein constituents of the mechanism responsible for the intestinal absorption of cholesterol.

Identification of a ligand-binding site in the Na+/bile acid cotransporting protein from rabbit ileum.

Reabsorption of bile acids occurs in the terminal ileum by a Na+-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4–4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6–7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na+/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56–67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.