TY - JOUR
T1 - High performance liquid chromatography of the 1, 25-dihydroxyvitamin D3 receptor
T2 - Its application toward resolution of receptor-monoclonal antibody complexes
AU - Pike, J. Wesley
AU - Kelly, Michael A.
AU - Haussler, Mark R.
N1 - Funding Information:
by NIH grant
PY - 1982/12/15
Y1 - 1982/12/15
N2 - The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.
AB - The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.
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U2 - 10.1016/0006-291X(82)92025-3
DO - 10.1016/0006-291X(82)92025-3
M3 - Article
C2 - 6297493
AN - SCOPUS:0020460627
SN - 0006-291X
VL - 109
SP - 902
EP - 907
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -