Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

Kramer, Werner; Girbig, Frank; Gutjahr, Ulrike; Leipe, Irina

doi:10.1016/0021-9673(90)85044-v

Cited by 29 publications

(16 citation statements)

References 20 publications

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“…The resulting pellet was resuspended in buffer and used immediately for experiments. After photoaffinity labelling, microsomes or particulate material were washed twice with the above buffer, proteins were precipitated [29] and analysed by SDS\PAGE [30,31]. Phase separation experiments to discriminate between integral and peripheral membrane proteins by alkaline extraction or treatment with non-ionic detergents were performed as described previously [30].…”

Section: Photoaffinity Labellingmentioning

confidence: 99%

“…Phase separation experiments to discriminate between integral and peripheral membrane proteins by alkaline extraction or treatment with non-ionic detergents were performed as described previously [30]. Radioactively labelled polypeptides were detected either by liquid-scintillation counting after the gels had been sliced into 2 mm pieces with subsequent hydrolysis of polypeptides with tissue solubilizer Biolute S or by fluorography as described [31].…”

Section: Photoaffinity Labellingmentioning

confidence: 99%

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Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

Kramer¹,

Burger²,

Arion

et al. 1999

Biochemical Journal

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The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [$H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [$H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3 % CHAPS retains the specific binding of T1 inhibitors ; photoaffinity labelling of such CHAPS-

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Section: Photoaffinity Labellingmentioning

confidence: 99%

Section: Photoaffinity Labellingmentioning

confidence: 99%

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

Kramer¹,

Burger²,

Arion

et al. 1999

Biochemical Journal

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“…The resulting supernatant was carefully removed and the pellet containing the liposomes was resuspended in 200 p1 buffer B with the aid of a 27-gauge syringe. The final liposome suspension was kept at 4'C and was used for further experiments within 4 h. For the preparation of proteoliposomes containing proteins from the flowthrough and the eluate of wheat germ lectin affinity chromatography, 10 mg brush border membrane vesicle protein was solubilized for 30 min at 4°C with 2 ml buffer C. After separation from nonsolubilized material by centrifugation, the clear supernatant containing solubilized brush border membrane proteins was submitted to affinity chromatography on wheat-germ-lectin -agarose or wheatgerm-lectin -Sepharose columns as described previously [19]. Samples (1 mg) of protein from the eluate, the flowthrough and solubilized brush border membrane proteins, were diluted to 1.2 ml with buffer C. The reconstitution of these different protein solutions, as well as of a pure lipid suspension, was performed as described above by mixing of 1.2 ml of these protein solutions or of buffer alone with 1.2 ml of the lipid suspension and subsequent gel filtration.…”

Section: Reconstitution Studiesmentioning

confidence: 99%

“…For the reconstitution of the purified binding protein for p-lactam antibiotics and oligopeptides of M, 127000, 1.2 ml of the elution peak maximum from Mono S ion-exchange chromatography [20] were adjusted to 200 mM n-octyl glucoside and submitted to the reconstitution procedure as described above. The lipid content in the different liposome preparations was determined using the Merckotest kit for total lipids.…”

Section: Reconstitution Studiesmentioning

confidence: 99%

“…The binding protein for b-lactam antibiotics and oligopeptides of M , 127000 was purified to more than 95% homogeneity by wheat-germ-lectin-chromatography at pH 7.4 [19] and subsequent FPLC cation-exchange chromatography on Mono S (HR 5/5) columns at pH4.5 [20]. The PI of the microheterogeneous glycoprotein was 5.38 -5.86 on two-dimensional gels using carbamolytes as standards.…”

Section: Migration (Cm)mentioning

confidence: 99%

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Intestinal absorption of β‐lactam antibiotics and oligopeptides

Kramer

Girbig

Gutjahr

et al. 1992

European Journal of Biochemistry

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The H+‐dependent uptake system responsible for the enteral absorption of oligopeptides and orally active β‐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 α‐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 β‐lactam antibiotics of Mr 127000 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 β‐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 β‐lactam antibiotics of Mr 127000 mediates the stereospecific and H+‐dependent transport of orally active β‐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).

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Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting

Maag

Alfredson

2008

Journal of Pharmaceutical Sciences

140

113

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Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

Cited by 29 publications

References 20 publications

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling

Intestinal absorption of β‐lactam antibiotics and oligopeptides

Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting

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