Characterization of fully active biotinylated parathyroid hormone analogs. Application to fluorescence-activated cell sorting of parathyroid hormone receptor bearing cells.
[Nle8,18,Tyr34]bPTH-(1-34)amide (NlePTH) was biotinylated using sulfosuccinimidyl 6-(biotinamido)hexanoate, in dimethyl sulfoxide, and the multiple resulting peptides peaks were separated by reverse-phase high performance liquid chromatography. Their biological activities were compared with those of NlePTH, the parent compound, in radioreceptor and cAMP accumulation bioassays using rat osteosarcoma 17/2.8 cells; the earliest two eluting products, bioPTH 1 and 2, were equipotent, a third, bioPTH 3, was only 10% as potent, and the remaining, later eluting derivatives all were less than 0.1% as active. Competitive avidin binding assays using [3H]biotin suggested that bioPTH 1 and 2 had a single biotin congener per molecule, while bioPTH 3 contained two biotin residues. Upon Edman degradation, bioPTH 1 contained biotin on the lysine at position 13 of NlePTH; bioPTH 2's biotin was on the lysine at position 26 (or 27) and bioPTH 3 had biotins on lysines at both positions 13 and 26 (or 27). Avidin tagged with 125I, peroxidase, or fluorescein isothiocyanate was detected on bone-derived cells which had been incubated initially with bioPTH 2 (1, 10, and 100 nM) for 4 h, but not when NlePTH (1 microM) was added with bioPTH 2. A fluorescence-activated cell sorter detected a symmetrical shift in fluorescence of bone-derived cells incubated with 10 nM of bioPTH 2 and 10 micrograms/ml fluorescein isothiocyanate-avidin. Addition of a 30-fold molar excess of NlePTH, or omission of bioPTH 2, completely reversed this fluorescence shift, and no shift in fluorescence was seen with cells lacking PTH receptors. This fully active, high affinity biotinylated PTH-derivative should prove useful in the study of PTH receptor-bearing cells.
Abou-Samra
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Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Freeman
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Jüppner
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Uneno
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D000818:Animals; D001360:Avidin / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D008239:Lysine / Q000032:analysis; D012516:Osteosarcoma / Q000378:metabolism; D010281:Parathyroid Hormone / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide; D001360:Avidin; D001710:Biotin; C061438:parathyroid hormone (1-34)amide, Nle(8), Nle(18), Tyr(34)-; D000242:Cyclic AMP; D008239:Lysine