Search

We confirmed our previous observation that duodenal Ca2+ absorption and serum 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels declined concurrently in old (24 months old) rats as compared to young (6 months old) rats. It is well known that 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) expresses its action after binding to specific receptor molecules. In this paper, we compared certain properties of rat duodenal 1,25-(OH)2D3 receptors from old and young animals. Receptor preparations were incubated with [3H]1,25-(OH)2D3 to quantitate the number of unoccupied and total receptor sites and showed that total and unoccupied receptor sites decreased by 22 and 16%, respectively in old rats. Endogenously occupied sites were reduced by 43% in duodenum of the old rat and, consequently, the percentage of receptor occupancy also declined. Age did not affect the dissociation constant (KD) of 1,25-(OH)2D3 from the receptor; the sedimentation coefficient (3.3 S) of the tritiated 1,25-(OH)2D3-receptor complex in sucrose density centrifugation; or its affinity for DNA. The data are consistent with the hypothesis that the age-related decline in Ca2+ absorption in the intestine may be due, in part, to the decrement in the circulating level of 1,25-(OH)2D and a reduction of intestinal 1,25-(OH)2D3 receptor occupancy status.

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and gamma-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na+ + K+1)-ATPase. However, the specific activity of (Na+ + K+)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na+ + K+)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na+ + K+)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5'-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN3 plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na+ + K+)-ATPase, be used as an enzyme "marker" for the renal basal-lateral membrane.

Phlorizin, phloretin, Tris and beta-methylglucoside are competitive inhibitors, with respect to the substrate trehalose, of purified renal trehalase. Mercuric chloride is a noncompetitive inhibitor. The active site of trehalase was examined further by multi-inhibition kinetic studies involving combinations of inhibitors. Phlorizin vs. phloretin and phlorizin vs. Tris were mutually non-competitive. In contrast, phloretin vs. Tris was mutually competitive. These findings suggest that the binding site of phlorizin to the enzyme differed from that of phloretin or Tris, and that phloretin and Tris might bind at a common site. These findings suggest a model in which trehalase has two binding sites at the substrate-binding site, a phlorizin (glucosyl) and a phloretin (phenyl) binding site, analogous to the model proposed previously for the glucose carrier. In addition, mercuric chloride vs. beta-methylglucoside was mutually competitive, although mercuric chloride and beta-methylglucoside, respectively, were noncompetitive and competitive inhibitors with respect to the substrate. Thus, it is suggested that the substrate binding and the SH-inhibitor binding sites are located very close to each other.

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na+ electrochemical gradient (extravesicular greater than intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K+, Li+ or choline+ gradient and was enhanced as extravesicular Na+ was increased from 10 mM to 100 mM. Dissipation of the Na+ gradient by the ionophore gramicidin D resulted in diminished Na+-stimulated glycine uptake. Na+-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na+-dependent glycine uptake over the range of amino acid concentrations from 25 microM to 10 mM demonstrated a single saturable transport system with apparent Km = 996 microM and Vmax = 348 pmol glycine/mg protein per min. Inhibition observed when the Na+-dependent uptake of 25 microM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids D-alanine, D-glutamic acid, and D-proline inhibited similarly to their L counterparts. Accelerative exchange of extravesicular [3H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with L-alanine, L-glutamic acid, or with L-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.

The specific activities of membrane-bound maltase (alpha-d-glucoside glucohydrolase, EC 3.2.1.20) in renal cortex homogenates and isolated brush border membranes of senescent rats decreased about 30% compared to the specific activities of the enzyme from young adult animals. The decline was gradual and concomitant with the aging process. When the enzymes from rats of 25 and 6 months of age were solubilized and purified to homogeneity the same decrement with age was found, 32.5 and 46.1 units/mg of protein, respectively. This finding suggests that the decrease in maltase activity with age results from an alteration in the enzyme per se, rather than from a change in the enzyme's membrane environment, which was reflected secondarily as a loss in activity. Recoveries of enzyme activity and protein and fold-purification were similar for young and old maltase, indicating that the age-related difference in specific activities of the pure enzymes was not due to the selective purification of an altered species of enzyme. The age-associated difference in activity was not attributable to the presence of proteolytic activity in the homogenate nor to the presence of an activator in the young or an inhibitor in the old kidney. The pure enzymes from young adult and aged animals did not differ in molecular weight, electrophoretic mobility, amino acid composition and Km value. Circular dichroism spectra revealed that both the young and old enzymes contained beta-structure. However, the old enzyme had more helical structure than did the young enzyme, suggesting a conformational alteration with age.