The two last eluted fractions (VIII and IX) represented the lower molecular mass fractions. Fraction IX had virtually no absorption at 280 nm ( Fig. 1a). Tricine SDS-PAGE analysis of fraction IX showed the presence check details of peptides with a molecular mass
estimated to be lower than 3 kDa. Subsequently, fraction IX was lyophilized and a new fractionation was performed using reverse phase HPLC. Among the amino acid sequences of the peptides found in fraction IX, only one matched with the features of the natriuretic peptide. This peptide was designated as TsNP (T. serrulatus Natriuretic Peptide) ( Fig. 1b). The molecular mass of TsNP was determined to be 2190.64 Da (as shown in Supplementary data). An isoelectric point of approximately 9.0 was this website calculated based on the N-terminal sequencing. The primary structure consisted of 21 amino acids, “KLSGCFGFKLDRIGTMSGLGC”, and included the cysteine residues that allowed the formation of a 17 amino acid ring held by a disulfide bridge. The results obtained by homology modeling of TsNP using the 1Q01 PDB structure are shown in Fig. 2. The quality of the model has been verified using PROCHECK (Laskowski et al., 1993). The overall G-factor is −0.22 and there are no residues in the disallowed regions of the Ramachandran plot. Multiple sequence alignments among the target (TsNP peptide) and reference sequences
were performed with the ClustalX program (Thompson et al., 1997) using the default parameters. The results can be found in Fig. 3. crotamiton The reference structures chosen for this alignment, with their respective accession numbers, follow: 1) ANPHs (human ANP – P01160) “SLRRSSCFGGRMDRIGAQSGLGCNSFRY”; 2) BNPHs (human BNP – P16860) “SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH”;
3) CNPHs (human – P23582) “GLSKGCFGLKLDRIGSMSGLGC”; 4) ANPRn (rat ANP – P01161) “SLRRSSCFGGRIDRIGAQSGLGCNSFRY”; 5) BNPRn (rat BNP – P13205) “SQDSAFRIQERLRNSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF”; 6) CNPRn (rat CNP – P55207) “GLSKGCFGLKLDRIGSMSGLGC”; and 7) DNPDa (Dendroaspis DNP – P28374) “EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA”. In isolated perfused rat kidney assay, both concentrations of TsNP (0.03 and 0.1 μg/mL) increased the perfusion pressure and urinary flow after 90 and 120 min of exposure. The glomerular filtration rate was augmented after 120 min at both concentrations. The higher TsNP concentration (0.1 μg/mL) also increased the GFR after 90 min. These results are shown in Fig. 4a–c. Renal vascular resistance was elevated only at 120 min in the group treated with TsNP at 0.1 μg/mL (RVR 120′ Cont. 5.38 ± 0.53; TsNP0.03 5.82 ± 0.48; TsNP0.1 6.71 ± 0.52* mmHg/mL g−1 min−1). The percentages of renal transport for sodium, potassium and chloride were decreased, as was the percentage of sodium proximal tubular transport, after treatment with TsNP 0.1 μg/mL (Table 2). Urinary cGMP concentration was elevated at both TsNP concentrations at 60 min (Cont. 8.83 ± 0.70; TsNP0.03 29.50 ± 5.