Development of a radioimmunoassay for quantitation of calregulin in bovine tissues.
Experimental conditions are described for a convenient and simple one-step method for radioimmunoassay (RIA) of the calcium binding protein calregulin [Waisman, D.M., Salimath, B.P., & Anderson, M.J. (1985) J. Biol. Chem. 260, 1652-1660]. The radioimmunoassay utilizes 125I-labeled calregulin and pansorbin cells (Staphylococcus aureus) coated with rabbit anti-calregulin antibody. Binding equilibrium is attained in 30 min, and the total time of the assay is 1 h. The assay is sensitive to about 30 fmol of calregulin. Calregulin was quantitated in various bovine tissue extracts and was detected in all tissues except erythrocytes. It was present in particularly high amounts in pancreas (540 micrograms/g of tissue), liver (375 micrograms/g of tissue), and testis (256 micrograms/g of tissue). While about 80% of the total tissue calregulin is soluble, about 20% of this protein was found to be associated with particulate fractions. Unmasking of particulate calregulin required the presence of nonionic detergent. Gel permeation chromatography of bovine brain 100 000 g supernatant in the presence or absence of calcium has resolved a single peak of calregulin by RIA. In addition, the distribution of calregulin in the soluble or particulate fraction of bovine brain was unaffected by the presence or absence of calcium during homogenization, suggesting that calregulin does not form either calcium-dependent or calcium-independent association with soluble or particulate proteins. These results identify calregulin as a major tissue Ca2+ binding protein.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis; D037282:Calreticulin; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D005347:Fibroblasts / Q000378:metabolism; D007457:Iodine Radioisotopes; D008099:Liver / Q000737:chemistry; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods; D012261:Ribonucleoproteins / Q000032:analysis* / Q000096:biosynthesis; D013462:Sulfur Radioisotopes; D014018:Tissue Distribution
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D007457:Iodine Radioisotopes; D012261:Ribonucleoproteins; D013462:Sulfur Radioisotopes; D008715:Methionine