Solubilization of 1,25-dihydroxyvitamin D3 receptor with pyridoxal 5'-phosphate from hen intestinal mucosa.

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5'-phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH)2D3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (greater than 90%) of the mucosal 1,25-(OH)2D3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5'-phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH)2D3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH)2D3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone.

Functional disability of rat splenocytes provoked to lipid peroxidation by cumene hydroperoxide.

Rat splenocytes were provoked to lipid peroxidation in a dose-dependent manner by cumene hydroperoxide. After exposure to cumene hydroperoxide, formation of high molecular weight protein, presumably through cross-linking of lower molecular weight protein, was stimulated in splenocytes as well as in erythrocyte ghosts. The mitogenic response to concanavalin A of splenocytes was remarkably depressed by addition of cumene hydroperoxide to cultures. This depression was due rather to failures of splenocytes in responding to concanavalin A than deactivation of concanavalin A molecules. It is notworthy that the viability of splenocytes was unaffected by cumene hydroperoxide under the culture conditions where the mitogenic response was depressed. The addition of alpha-tocopherol or thiourea could block the depression of mitogenic response by cumene hydroperoxide, indicating that the depressed response to concanavalin A was related to radical formation. Overall evidence suggests that the function of immunocompetent cells can be depressed through lipid peroxidation-associated mechanisms without suffering from lethal damage.

Membrane-associated vitamin D-induced calcium-binding protein (CaBP): quantification by a radioimmunoassay and evidence for a specific CaBP in purified intestinal brush borders.

The vitamin D-induced intestinal calcium-binding protein (CaBP) was quantitated in membranous components of the intestinal mucosa by a specific and sensitive RIA. Inclusion of detergent (Triton X-100) in extraction buffer and in the RIA system was required to release and measure membrane-associated CaBP. Purified brush borders were shown to contain CaBP with a specific activity (micrograms per mg protein) about 12% of that in the total homogenate. By transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose blots electrophoretically, CaBP was immunologically detected in brush borders from vitamin D3-treated chicks, but not in those from vitamin D3-deficient chicks. CaBP was also detected in isolated brush border membrane vesicles by the gel electrophoresis-blot transfer technique. Brush border CaBP was inaccessible to proteolytic hydrolysis by trypsin unless trypsinized in the presence of detergent. CaBP-binding substances were found to be present in purified brush borders, using the gel overlay technique. A specific binding protein with a mol wt in the range of 50,000-70,000 daltons was identified, as well as an avid CaBP binder at less than 14,000 mol wt. These observations provide evidence for the association of a significant fraction of total intestinal CaBP with brush borders in vivo, which might have physiological relevance.

Intestinal absorption of dinitrophenyl-lysine and effect of immunization with dinitrophenylated bovine serum albumin.

The intestinal absorption of dinitrophenyl-lysine (DNP-lys) was studied with a special interest on the role of the immune system in the absorption of small molecules which are recognized as nonself. [3H]-DNP-lys was rapidly absorbed by ligated intestinal loops in situ via a saturable and unique route. When [3H]-DNP-lys was preincubated with the immune serum obtained from rats immunized with dinitrophenylated bovine serum albumin (DNP-BSA), the [3H]-DNP-lys absorption was depressed. The absorption of [3H]-DNP-lys in DNP-BSA-immunized rats was depressed compared to the control. The results obtained suggest that the immune system play a role in avoiding the absorption of small molecules with antigenicity.

Effect of pyridoxal 5'-phosphate on the 1,25-dihydroxyvitamin D3 receptor system.

The effect of pyridoxal 5'-phosphate on the 1,25-dihydroxyvitamin D3 receptor system has been studied by using pig intestinal chromatin. Pyridoxal 5'-phosphate did not affect the binding of 1,25-dihydroxyvitamin D3 to its receptor extracted from chromatin with hypertonic KCl, although in the presence of pyridoxal 5'-phosphate 1,25-dihydroxyvitamin D3-receptor complexes were not readily precipitated with polyethylene glycol. In contrast, pyridoxal 5'-phosphate showed a potency to dissociate the 1,25-dihydroxyvitamin D3 receptor from chromatin in a dose-dependent manner. A low concentration of pyridoxal 5'-phosphate was as effective as hypertonic KCl in dissociating the receptor from chromatin, while pyridoxine, p-nitrophenyl phosphate, or inorganic phosphate was much less effective. These observations suggest the inhibitory effect of pyridoxal 5'-phosphate on the recognition of 1,25-dihydroxyvitamin D3 by its receptor system.

Biliary immunoglobulins in protein-energy malnourished rats.

The effect of protein-energy malnutrition on biliary immunoglobulins was investigated in rats fed isocaloric diets containing 0.5%, 5%, and 18% casein, respectively. Growth was severely retarded in rats fed 0.5% casein diet and moderately in rats fed 5% casein diet, and these groups had decreases in serum albumin and total protein levels. Since the energy intake was low in rats fed protein-insufficient diets, the nutritional status was defined not as protein malnutrition but protein-energy malnutrition. Depression of systemic immune functions in protein-energy malnourished rats were demonstrated by serum IgG and IgA levels, and antibody responses to dinitrophenylated bovine gamma globulin, a T-cell dependent antigen. The depressed systemic immune functions observed in those rats were suggested to be caused by thymic atrophy. IgA levels in bile were much higher in all groups than IgG levels. IgG levels decreased in rats fed 0.5% casein diet but not in rats fed 5% casein diet, while IgA levels decreased in rats fed 5% and 0.5% casein diet relating to casein levels. The ratios of IgA to IgG in bile also decreased in rats fed protein-insufficient diets. By sucrose density gradient centrifugation secretory IgA levels in bile were shown to decrease in rats fed 0.5% casein diet, suggesting that the secretion of IgA by hepatic parenchymal cells is depressed in the protein-energy malnourished rats.

Interaction of 1 alpha, 24-dihydroxyvitamin D3 with the chick intestinal 1 alpha, 25-dihydroxyvitamin D3 receptor system.

The interaction of 1 alpha, 24-dihydroxyvitamin D3 with the 1 alpha, 25-dihydroxyvitamin D3 receptor system was studied in chick intestinal mucosa. The competitive receptor binding assay indicated that 1 alpha, 24-dihydroxyvitamin D3 bound to a cytosol 1 alpha, 25-dihydroxyvitamin D3 receptor with a relatively high affinity compared with other vitamin D3 analogs. The R-isomer of 1 alpha, 24-dihydroxyvitamin D3 revealed higher affinity for the receptor than the S-isomer. In the reconstituted cytosol-chromatin system, bith 1 alpha, 24(R)-dihydroxy[3H]-vitamin D3 and 1 alpha, 24(S)-dihydroxy-[3H]-vitamin D3 specifically associated with chromatin via a temperature-dependent process. The association of 1 alpha, 24-dihydroxy-[3H]-vitamin D3 with chromatin was reduced in the presence of competing unlabeled 1 alpha, 25-dihydroxyvitamin D3. Furthermore, the chromatin-associated 1 alpha, 24-dihydroxy-[3H]-vitamin D3 was dissociated by a high concentration of KCl, likewise 1 alpha, 25-dihydroxy-[3H]-vitamin D3. From these results, it is strongly indicated that 1 alpha, 24-dihydroxyvitaim D3 is recognized by cytosol 1 alpha, 25-dihydroxyvitamin D3 receptor as an analog of 1 alpha, 25-dihydroxyvitamin D3 and associates with chromatin by the same mechanism as 1 alpha, 25-dihydroxyvitamin D3.

Some characteristics of cytosol binding protein for 1alpha,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in rat intestinal mucosa.

Cytosol binding protein for 1alpha,25-(OH)2D3 and 25-OHD3 was demonstrated in rat intestinal mucosa using sucrose density gradient ultracentrifugation analysis, Sephadex G-200 column chromatography and polyacrylamide disc gel electrophoresis. The binding protein has a sedimentation constant of 5-6S, a molecular weight of 100,000-120,000 daltons and a mobility in electrophoresis of Rf 0.42. Further analysis of binding behavior by DEAE-cellulose filter assays revealed that the bindig protein had a higher affinity for 25-OHD3 than for 1alpha,25-(OH)2D3, and that 1alpha,25-(OH)2D3 competed with 25-OHD3 for the same binding site on the protein. The apparent dissociation constant for 1alpha,25-(OH)2D3 and 25-OHD3 was 9.2 X 10(-9) M and 1.5 X 10(-9) M, respectively. The binding capacity was 3.1 pmoles/mg protein for both 1alpha,25-(OH)2D3 and 25-OHD3. The order of binding affinity was 25-OHD3 greater than 1alpha,25-(OH)2D3 greater than D3 = 1alpha-OHD3.

Development of 1,25-dihydroxycholecalciferol receptor in the duodenal cytosol of chick embryo.

The development of 1,25-(OH)2D3 receptor in the duodenal cytosol of chick embryo was studied by the sucrose density gradient analysis. The binding profile for 1,25-(OH)2D3 in the cytosol of vitamin D-deficient chick duodenum on the sucrose density gradient revealed 3 binding components, and the sedimentation constant was estimated as 2.5, 3.5 and 5.5S respectively. The 3.5S binding component has high affinity and low capacity for 1,25-(OH)2D3 and is thought to be 1,25-(OH)2D3 receptor. During the development of chick embryo, the 3.5S binding component was not detected in 13-day embryonic duodenum, it appeared on 15th day of incubation and then gradually increased to the level of vitamin D-deficient chick on 19th day of incubation. The 5.5S binding component was specific for 25-OH-D3 and it was found even in 13-day embryo, but it did not show any significant change during development. On the other hand, the 2.5S component was not specific for either 1,25-(OH)2D3 or 25-OH-D3. However, it was main binding component in early stages of development and decreased during development. From these results, it is suggested that the receptor for 1,25-(OH)2D3 is available a few days before hatching and the inability to produce CaBP in the duodenum of chick embryo could not be ascribed to the absence of the receptor.

Synthesis and biological activity of 5,6-trans-vitamin D3 in anephric rats.

A modified procedure for the purification of 5,6-trans-D3 from a reaction mixture of vitamin D3 with iodine and its biological activity in sham-operated and anephric rats are described. When a solution of vitamin D3 in n-hexane was reacted with small quantities of iodine under influence of visible light, the reaction mixture gave four or five spots, including vitamin D3 and 5,6-trans-D3, on a thin layer chromatogram. After purifying the mixture by alumina column chromatography, a colorless oil from the separated 5,6-trans-D3 fractions was crystallized from n-hexane and snow white crystalline 5,6-trans-D3 was obtained. The purification method was thought to be better than the previously reported methods (14-16) because it gave good yield without the procedure of esterification using expensive p-phenylazobenzoyl chloride. A dose of 25 mug of 5,6-trans-D3 obtained thus gave significant elevation on intestinal calcium transport in both sham-operated and anephric rats, whereas the dose did not give positive effects on serum calcium levels in anephric rats. Serum phosphorous levels were extremely elevated by nephrectomy, but in both sham-operated and anephric rats they were unaffected by the administration of 25 myg of 5,6-trans-D3.