Cell-free translational analysis of messenger ribonucleic acid coding for vitamin D-dependent rat renal calcium-binding protein.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.

Vitamin D-dependent rat renal calcium-binding protein: development of a radioimmunoassay, tissue distribution, and immunologic identification.

A sensitive double antibody RIA has been developed for the 28,000 mol wt rat renal vitamin D-dependent calcium-binding protein. Using this assay, concentrations of calcium-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraassay variability, 5.0%) and reproductible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CaBP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 +/- 1.0 (mean +/- SEM) micrograms/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 +/- 0.3 micrograms/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 +/- 5.1 micrograms/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 +/- 0.1 and 27.3 +/- 2.1 micrograms/mg protein, respectively). When extracts of rat kidney and brain and human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney, and human cerebellum on Sephadex G-100 revealed immunoreactivity and calcium-binding activity in the 28,000 mol wt region similar to rat kidney.

Vitamin D-dependent calcium-binding protein in rat kidney. Purification and physiocochemical and immunological characterization.

A vitamin D-dependent Mr = 28,000 calcium-binding protein (CaBP) has been isolated from rat kidney. Rat renal CaBP was purified from heat-treated post-mitochondrial supernatants by gel filtration on Sephadex G-100 followed by preparative gel electrophoresis. The specific properties and characteristics of the protein were examined. Rat renal CaBP was found to have a pI of 4.8 and showed increased electrophoretic mobility during polyacrylamide gel electrophoresis in the presence of 1 mM EDTA. Amino acid analysis of renal CaBP revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, cysteine, and tyrosine, similar but not identical to chick intestinal CaBP. Circular dichroism studies indicated that the alpha-helical content of renal CaBP was of the order of 31% and was changed to a minor degree by the addition of calcium. A study of the thermal stability indicated that renal CaBP is heat-stable up to 75 degrees C. Binding studies utilizing the technique of equilibrium dialysis established a dissociation constant of 2.1 X 10(-6) M and binding sites with a capacity of approximately 4 mol Ca2+/mol of CaBP. Immunologically, using Ouchterlony immunodiffusion, a precipitin line which joined with total coalescence with that due to purified renal CaBP was observed with extracts of rat brain and kidney but not with extracts of rat parathyroid, skeletal muscle, myocardium, bone, pancreas, intestine, and liver. In summary, these studies present the first purification and characterization of vitamin D-dependent rat renal calcium-binding protein. The antibody as well as the protein will be useful for the development of a radioimmunoassay and for the determination of the molecular mechanisms of induction of renal calcium-binding protein.

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein.

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorus or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca2+ bound protein/Ca2+ bound resin per mg protein) in the 28,000 Mr region was found to increase four fold in rats fed the low phosphorus diet and two fold in rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 Mr fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.

Enhanced insulin secretion and calcium uptake by Zucker "fatty" rat islets.

The relationship of the net uptake of calcium to insulin secretion by pancreatic islets isolated from Zucker "fatty" rats and their lean counterparts was studied. Islets from "fatty" rats secreted 1.5 to 3 times as much insulin as did the lean rat islets over a glucose concentration range of 0 to 27.7 mM. Over the same glucose concentration range, calcium accumulation was 2-fold greater in islets from the "fatty" rats than from the others. Both insulin secretion and calcium uptake were 2 to 3 fold greater for islets from the "fatty" rats than those from the lean animals over an extracellular calcium concentration range of up to 5 mEq/L. The data indicate that for islets isolated from Zucker "fatty" rats insulin hypersecretion in response to glucose and extra-cellular calcium is associated with enhanced calcium accumulation.