Smooth-hound

Abstract: Trypsin from the intestine of smooth hound (Mustelus mustelus) was purified by fractionation with ammonium sulfate, Sephadex G-75 gel filtration, and DEAE-cellulose ion exchange chromatography, with a 65-fold increase in specific activity and 15% recovery. The molecular weight of the purified trypsin was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed esterase-specific activity on N(alpha)-p-tosyl-L-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nalpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 degrees C, respectively, using TAME as a substrate. The enzyme was extremely stable in the pH range of 7.0-9.0 and highly stable up to 40 degrees C after 1 h of incubation. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK), specific inhibitors for trypsin. In addition, smooth hound trypsin showed higher proteolytic activity at high NaCl concentration, demonstrating its potential for protein hydrolysis at high salt content. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECKPHSQ. This sequence showed high homology with trypsins from marine vertebrates and invertebrates. Purified trypsin had a Michaelis-Menten constant (K(m)) and catalytic constant (K(cat)) of 0.387 +/- 0.02 mM and 2.62 +/- 0.11 s(-1), respectively, when BAPNA was used as a substrate. For the hydrolysis of TAME, K(m) and K(cat) were 0.156 +/- 0.01 mM and 59.15 +/- 2.2 s(-1), respectively.

Abstract: In this study, smooth hound protein hydrolysates (SHPHs), obtained by treatment with various gastrointestinal proteases, were analyzed for their angiotensin I-converting enzyme (ACE) inhibitory activities. Protein hydrolysates were obtained by treatment with crude alkaline enzyme extract, low molecular weight (LMW) alkaline protease, trypsin-like protease and pepsin from Mustelus mustelus, and bovine trypsin. All hydrolysates exhibited inhibitory activity toward ACE. Hydrolysate generated with alkaline protease extract displayed the highest ACE inhibitory activity, and the higher inhibition activity (82.6% at 2 mg/mL) was obtained with a hydrolysis degree of 18.8%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (P1–P5). ACE inhibitory activities of all fractions were assayed, and P3 was found to display a high ACE inhibitory activity (62.24% at 1 mg/mL). P3 was then fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and ten fractions of ACE inhibitors were found (F1–F10). Sub-fraction F3 showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 100 μg/mL. The amino acid sequence of peptide F3 was determined by ESI/MS and ESI–MS/MS as Ala-Gly-Ser, and the IC50 value for ACE inhibitory activity was 0.13 ± 0.03 mg/mL. Further, purified peptide F3 maintained inhibitory activity even after in vitro digestion with gastrointestinal proteases in order to demonstrate gastrointestinal stability digestion to enable oral application. These results indicate that smooth hound protein hydrolysate possesses potent antihypertensive activity.

Abstract: Five species of smooth-hound sharks genus Mustelus (Family Triakidae) are know in the western South Atlantic, as follows: Mustelus canis (Mitchell 1815); Mustelus fasciatus (Garman 1913); Mustelus higmani Springer & Lowe 1963; Mustelus norrisi Springer 1939; and Mustelus schmitti Springer 1939. In the present paper, new data on the anatomy, morphometrics and meristic characters are given. Taxonomic aspects and comparison between the species are discussed. Most general body morphologic measurements and proportions are useless as a tool for species identification, since many of them show remarkable intraspecific variations. Head proportions and structures related seem to be a more adequate procedure to identify the species of Mustelus. The labial folds proportions, internasal distance and orbit diameter were the most useful character to separate the western South Atlantic species. The buccopharingeal pattern of denticles as well as tooth counts not was useful to distinguish the Mustelus species from western South Atlantic adequately, due great intraspecific variation.