Abstract
A protein capable of stimulating bone resorption in vitro has been purified approximately 1250-fold from cancer ascites fluid. Purification was accomplished employing successive fractionation with ammonium sulfate, ion exchange, and Cibacron blue affinity chromatography, isoelectric focusing, and selective adsorption on hydroxylapatite. The bone-resorptive protein obtained by this procedure appeared homogeneous in polyacrylamide gels at pH 9.5, migrating with the mobility of an alpha 2-globulin, and in sodium dodecyl sulfate polyacrylamide gels from which an apparent molecular weight of 43,000 was calculated. The amino acid composition of the bone-resorptive protein distinguished itself by the absence of methionine and by its relatively high content of glycine (17%) and proline (11%). Furthermore, the protein possesses a single NH2-terminal amino acid residue (glycine). The ascites protein was found to contain 19% carbohydrate by weight including a high content of sialic acid (15 residues/mol) as compared to the other sugars (27 residues/mol). As to its biological properties, the homogeneous ascites glycoprotein proved to be as potent as parathyroid hormone in its ability to stimulate bone resorption in vitro.
Highlights
The specific activity of AMs-50 (TaIb)lweas increased approximately 4-fold over the starting materials, and most (74%) of the activity was recovered in this protein fraction, 20% of the activity precipitating during dialysis and equilibration (AMs ppt, Table I)
The dose-response curve for the HA-1 preparation demonstrates that,while 1 pg of HA-l/ml of culture medium did not stimulate significant resorption over controls, there was a rapid increase in the release of 45Ca2+from 2 to 4 pg/ml
The results of calvarial explants cultured in the presence of indomethacin (Table IV) demonstrate that the bone-resorptive activity of the isolated as-glycoprotein is not inhibited by this compound
Summary
Localized osteolytic lesions often occur in patients with a wide variety of neoplasms. The cultures were incubated for 3 days to allow for the removal of most of the readily exchangeable 45Ca" from the explants in order that the subsequent release of 45Ca'+from the explant would be due primarily to the active resorption of bone After this 3-day incubation, the medium was removed and one half-calvaria received new medium containing the protein fraction to be tested while its paired halfcalvaria received medium alone. One "bone-resorbing unit" of activity was defined as the quantity of material (micrograms per m l ) needed to produce a 50% increase in 45Ca2+release in treated over control bone explants (treated/control ratio = 1.5) The reliability of this assay has previo45uCsalyrbeeleenasedetrmeoatnesdtr/catoendtro(2l7,ra2t8io).sItofshpoauirlds aolfsohablef-cnaoltveadritahawthtohsee medium contains either no protein or inactive preparations is very close to unity. In this assay system, one unit of activity is produced by a dose of 0.5 pg/ml of PTH
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