Abstract
Transport of labeled glycyl-L-proline has been shown to occur with highly purified brush border membrane vesicles from the epithelial cells of rabbit small intestine and renal cortex. With 1-min incubation, transport occurs mainly as the intact dipeptide since less than 10% of the dipeptide in the medium is hydrolyzed within the period. The properties of the dipeptide transport system are similar in both small intestinal and renal brush border membrane vesicles. The steady state transport varies inversely with medium osmolarity. Extrapolation to infinite medium osmolarity indicates that transport occurs predominantly into an osmotically reactive intravesicular space rather than binding to the membranes. The affinity constants (Kt) for glycyl-L-proline transport in small intestinal and renal brush border membrane vesicles are comparable (0.9 mM in intestine and 1.1 mM in kidney). Under conditions in which presence of a Na+ gradient between external and intravesicular media stimulated L-alanine transport, glycyl-L-proline transport remains unaffected. Other dipeptides strongly inhibit the transport of glycyl-L-proline but amino acids have no effect. The selective inhibition of glycyl-L-proline transport by other dipeptides is observed in the presence as well as in the absence of a Na+ gradient. Harmaline inhibits Na+-stimulated L-alanine transport but it has no effect on glycyl-L-proline transport even in the presence of Na+. In these respects, dipeptide transport seems to differ from amino acid transport. It is proposed that the Na+ gradient hypothesis of sugar and amino acid transport is not applicable for dipeptide transport. These data provide additional evidence for the distinct nature of amino acid and dipeptide transport systems.
Highlights
Vesicles from the epithelial cells of rabbit small intes- proline by small intestine was only partially dependent on tine and renal cortexW. ith I-min incubation, transport Na’ since an appreciable transport of the dipeptide remained occurs mainly as the intact dipeptide since less than after Na’ replacement and this could not be attributed to
Extrapolation to infinite medium osmolarity indicates that transport occurs predominantly into an osmotically reactive intravesicular space rather thabninding to themembranes
L-proline transport in small intestinal and renal brush Recently, isolated brushbordermembrane vesicles both border membrane vesicles are comparable (0.9 mM in from small intestine and renal cortex have been used by a intestine and 1.1 mM in kidney)
Summary
Preparation of BrushBorderMembrane Vesicles-The brush lished various characteristics of dipeptide transport systems, border membranevesicles were prepared from rabbit small intestine yet one problem remains unresolved. Rubino et al [10]showed the presence of Na’ increased V,,,, for glycyl-L-proline transportwithout altering K,,whereas theeffect of Na’ on glycine transport was scraped off and homogenized in 30 volumes (v/w) of ice-cold 50 mM mannitol/2 mM Tris-HC1 buffer, pH 7.0, for 2 min using a Waring Blendor. Proline was studied, the osmolarity of the buffer was adjusted to 300 amino acids (A grade) were from Calbiochem. Uptakeintobrushbordermembrane [l-'4C]Glycyl-~-proline(specific radioactivity, mCi/mmol) was vesicles was initiated by obtained from the Ra&ochemical Center, Amersham, England, and addition of 50p1 of the membranesuspension containing 0.6 to 0.8 mg ~-[U-"C]alanine (specific radioactivity, 160 mCi/mmol) was from of membrane protein to 100 pl of uptake buffer. At the endof the incubation period, uptake was stopped by adding 2 ml of ice-cold 154 mM NaCl
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