Calcium-Dependent Activation of Guanylate Cyclase by S100b

Calcium concentration in the dark-adapted retinal rod outer segment is in the 200 to 600 nM range, and the guanylate cyclase of rod outer segments is thought to be activated in response to a fall in calcium concentration triggered by light. Calcium-binding proteins that mediate such activation, i.e., activation in the absence of or presence of low nanomolar calcium concentrations, have been identified and termed GCAPs (Guanaylate Cyclase Activating Proteins). In the course of our search for GCAP-like proteins in bovine retina, we isolated a protein fraction that stimulated rod outer segment cyclase activity at calcium concentrations higher than those in dark-adapted rod outer segments. We purified the protein responsible for this calcium-dependent stimulation of cyclase activity and found it to be of 6-7 kDa molecular weight as judged by electrophoresis under denaturing conditions and about 40 kDa by gel filtration analysis. Maximum stimulation of cyclase activity was observed at 3-4 micromolar concentration of the protein. It required about 1.5 micromolar free calcium concentration for half-maximal activation of the enzyme. Partial amino acid sequencing of peptide fragments of the activator suggested that the protein was identical with S100b, a previously described calcium-binding protein. Further characterization with antibody specific for S100b supported this possibility. However, the protein isolated in our laboratory and termed CD-GCAP (Calcium-Dependent Guanylate Cyclase Activator Protein) was found to differ significantly from commercially available S100b in the magnitude and calcium dependence of cyclase activation. It was also found to be inactivated by hydroxylamine while S100b was resistant. Investigation into these differences showed that purification methods had a significant influence on the properties of the activator, producing a less active (S 100b) or more active (CD-GCAP) protein, but that it was, otherwise, one and the same protein. We conclude from this study that rod outer segment guanylate cyclase, unlike any cyclase known so far, is capable of activation by two different types of calcium-binding proteins, one that activates in response to a decrease in calcium concentration, and the other, described here, which activates in response to an increase in calcium-concentration. We hypothesize that this cyclase and others like it will be colocalized with one or the other type of activator depending upon the physiological requirement, i.e., activation in response to decreasing or increasing calcium concentration.