STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF RETINAL CALCIUM-DEPENDENT GUANYLATE-CYCLASE ACTIVATOR PROTEIN (CD-GCAP) - IDENTITY WITH S100-BETA PROTEIN

Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a l ow-molecular-weight retinal calcium-binding protein which activates ro d outer segment guanylate cyclase (ROS-GC) in a calcium-dependent mann er. This investigation was undertaken to determine the protein's struc ture and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed t hat CD-GCAP is identical to S100 beta, another law-molecular-weight ca lcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100 beta beta or dimer of S100 beta), also activates ROS-GC but that the V-max of a ctivated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-5) vs 1.5 x 10(-6) M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S 100b, both proteins were treated with I M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD- GCAP could not be a modified form of S100b. Hydroxylamine also broke d own CD-GCAP into smaller fragments while leaving S100b intact. It ther efore appeared that in spite of identical primary structures, the conf ormations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, whi ch were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 de grees C in 5 mM Ca, while S100b purification included zinc affinity ch romatography. To test the influence of these treatments on the propert ies of the proteins, CD-GCAP was subjected to zinc affinity chromatogr aphy and purified as S100b (CD-GCAP-->S100b) and S100b was heated in C a and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calci um-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP S100b is identical to S100b and that S100b-->CD-GCAP is identi cal to CD-GCAP. Taken together the results demonstrate that CD-GCAP an d S100b are one and the same protein and that their functional differe nces are due to different interconvertible conformational states.