title: Structural and functional characterization of retinal calcium-dependent guanylate cyclase activator protein (CD-GCAP): identity with S100beta protein.
abstract: Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated 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 S100b, both proteins were treated with 1 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 down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.
authors.affiliation: Eye Research Institute, Oakland University, Rochester, Michigan 48309, USA.
authors.forename: N
authors.initials: N
authors.lastname: Pozdnyakov
authors.forename: R
authors.initials: R
authors.lastname: Goraczniak
authors.forename: A
authors.initials: A
authors.lastname: Margulis
authors.forename: T
authors.initials: T
authors.lastname: Duda
authors.forename: R K
authors.initials: RK
authors.lastname: Sharma
authors.forename: A
authors.initials: A
authors.lastname: Yoshida
authors.forename: A
authors.initials: A
authors.lastname: Sitaramayya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D002417:Cattle; D003001:Cloning, Molecular; D003429:Cross Reactions; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D019811:Hydroxylamine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012160:Retina / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA
chemical_list: D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D019811:Hydroxylamine; D006162:Guanylate Cyclase; D002118:Calcium