The reactivity of recombinant and
tumor-derived preparations of
oncomodulin toward 5,5'-dithiobis-(2-nitrobenzoate) (
Ellman's reagent) and
dansylaziridine was investigated. In contrast to previously published data (Mutus, B., Palmer, E. J., and MacManus, J. P. (1988) Biochemistry 27, 5615-5622), the
apoprotein was observed to react far more rapidly than the
calcium-bound form with
Ellman's reagent. Attempts to quantitatively label the native
protein with
dansylaziridine met with little success, either with the
metal-free or
calcium-bound forms. In neither case did the extent of modification approach the level observed with the
sodium dodecyl sulfate-denatured form of the
protein. These results suggest that access to the sulfhydryl group of Cys-18 is severely restricted in the native
protein, particularly when the high affinity ion-binding sites are occupied. Consistent with these observations, prolonged incubation of native
oncomodulin at room temperature in the absence of
reductant did not result in the generation of
disulfide-linked dimers, either in the presence or absence of Ca2+. Interestingly, however, Cu2+ ion was observed to facilitate the apparent dimerization of
oncomodulin. This reaction, which occurs more rapidly with the Ca2(+)-free form of the
protein, affords material with the expected electrophoretic mobility. However, in contrast with the results of Mutus et al., dimeric
oncomodulin prepared in this manner fails to stimulate bovine heart
cAMP phosphodiesterase.