Since the khd segment is the least preserved and least preserved, the initial intuition was that the khd segment, aa737-766, could represent the δ-modulated neurocalcin domain. This possibility was tested by the use of a soluble construction, L806-T986 (Figure 2A). This construction lacked the segment khd aa733-766 and especially the dd. The choice of this construction was based on the idea that it would separate the catalytic domain from the domain that houses the neurocalcin δ-modulated domain. The decision to assign the L806-T986 limit to the Catd was to retain this domain among all members of the membrane-guanylate-cyclase family, and there is even more conservation among members of the ROS-GC subfamily, 92% with ROS-GC2 and 85% with ONE-GC. As the catd`s basic area (catcd) is not clearly defined (44, 45) in order not to disturb its functional integrity as a preventive measure, the design of the L806-T986 construction was such that its boundary extended to the cte domain. This strategy was based on original information from the Guanylatcyclase surface receiver subfamily, in which the catalytic domain extended and ended at the end of Terminal C. As the L806-T986 fragment did not contain dd, it was expected that this area would be monomenaire and not to have intrinsic and regulatory activity. Previous studies have shown that dd is mandatory for signal transduction activity of all membrane guanyatcyclases (44, 46, 47). The predicted function of dd is to form the contact points for the homodimization of the catalytic module and to move this module from inactive to active. The crystalline structure of Adenylatcyclase II (PDB ID 1AZS; 52) [AC2 C2 domain homodimer board was the model of the 1AWL model (50)”. The program used in this study was Phyre. It contains the secondary structure with the Aa sequenced profiles of the protein.
The model`s monomer is shown in Figure 8A; it is called a modified catcd model (modified model) to distinguish it from the 1AWL model. The modified model includes the M816-L1006 area of the catalytic domain; in particular, the region does not include the ROS-GC1 pivotal region (dd in Figure 2A). To assess Catcd`s oligomal status, a gel filtration analysis was performed. The purified protein was loaded onto a Superdex 75 column, and the treated fractions were controlled by absorption at 280 nm. A single peak was observed with a retention time of 19.4 min (Figure 2B). Based on calibration with molecular mass standards (Figure 2B, inclusion), this peak corresponded to the 50 kDa protein, suggesting that Catcd exists as dimer in its isolated form. GCAPs and S100B stimulated ROS-GC1 activity, 6, 5.5 and 9 times for GCAP1, GCAP2 and S100B, but had no effect on isolated Catcd activity (Figure 6). Therefore, the modular neurocalcin sites δ, GCAP1, GCAP2 and S100B are different in THE ROS-GC1 and are totally independent of each other`s activity.
The neurocalcin interaction point δ with ROS-GC1 is located in the field of ROS-GC1 G817-Y965. (A) Schematic representation of the soluble fragments of ROS-GC1. The following abbreviations refer to the predicted domains: dd, Dimerization Domain; katad, cyclase catalytic domain; catcd; The basic catalytic domain; Cte, extension of Terminal C. The numbers displayed correspond to the mature protein. The specific activity of the fragments is shown in the right column ([A]). (B) Analysis of the catcd domain cleaned by gel filtration. The ROS-GC1 catcd domain was used in E. coli and cleaned.
The purified protein was loaded onto a Superdex 75 gel filtration column and analyzed on an Akta FPLC system. The loading and evasive equipment was in the buffer with 220 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The elition was controlled by absorption at 280 nm. Only one peak was observed with a retention time of 19.4 min, or a height of 50 kDa.