Reactivity of cysteine 18 in oncomodulin.
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.
Clayshulte
T M
TM
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Taylor
D F
DF
Henzl
M T
MT
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002417:Cattle; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature
D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C034733:oncomodulin; D003300:Copper; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D003545:Cysteine; D002118:Calcium