Antiproliferative Potential of Olneya tesota PF2 Lectin in Human Acute Monocytic Leukemia Cells.

Acute monocytic leukemia is a type of myeloid leukemia that develops in monocytes. The current clinical therapies for leukemia are unsatisfactory due to their side effects and nonspecificity toward target cells. Some lectins display antitumor activity and may specifically recognize cancer cells by binding to carbohydrate structures on their surface. Therefore, this study evaluated the response of the human monocytic leukemia cell lines THP-1 to the Olneya tesota PF2 lectin. The induction of apoptosis and reactive oxygen species production in PF2-treated cells was evaluated by flow cytometry, and the lectin-THP-1 cell interaction and mitochondrial membrane potential were evaluated by confocal fluorescence microscopy. PF2 genotoxicity was evaluated by DNA fragmentation analysis via gel electrophoresis. The results showed that PF2 binds to THP-1 cells, triggers apoptosis and DNA degradation, changes the mitochondrial membrane potential, and increases reactive oxygen species levels in PF2-treated THP-1 cells. These results suggest the potential use of PF2 for developing alternative anticancer treatments with enhanced specificity.

Chymotrypsin isolation from jumbo squid (Dosidicus gigas) hepatopancreas: Partial characterization and effect on muscle collagen.

Chymotrypsin was purified from jumbo squid hepatopancreas (HP) with 2.4-fold and yield 1.9%, and characterized with a molecular weight of 31 kDa, as estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin effect over collagen extracted from the mantle, fins and arms of the jumbo squid was evaluated. The enzyme exhibited the maximum activity at pH 7 and 65°C using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as a substrate and it was identified using the specific inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and phenyl methyl sulfonyl fluoride (PMSF), showing residual activities of 6% and 0%, respectively. Furthermore, high activity was observed in the pH range of 4.0 to 8.0. Purified enzyme showed a moderate in vitro activity using muscle collagen as a substrate. Although further research is needed, the results suggest that the enzyme has a potential application where acidic or slightly alkaline conditions are needed.

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization.

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.

Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991.

Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 µmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.