A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Antileishmanial effects of Crotalaria spectabilis Roth aqueous extracts on Leishmania amazonensis.

Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.

Study of the proteolytic activity of the tropical legume Crotalaria spectabilis.

The characterization of legume proteases contributes to the understanding of the physiology of plants and their interaction with the environment. Thirteen extracts from various parts of Crotalaria spectabilis were made using different extraction systems. The highest protein content was found in seeds, and the most pronounced proteolytic activity was observed in leaf extracts, with an optimal pH value in the alkaline range. Proteases in extracts from roots, stems, and flowers were active in various pH ranges. Proteases in all extracts were maximally active between 30 degrees C and 60 degrees C and were thermostable (24 h, 60 degrees C). Hemoglobin, bovine serum albumin, casein, and gelatin were hydrolyzed by C. spectabilis extracts in different ways. The highest serine protease activity was found in leaves. Seeds contained high levels of serine proteases and low levels of cysteine proteases. Flowers, roots, and stems contained different levels of serine, aspartic, and metalloproteases, respectively. The proteolytic activities in extracts were modulated by cations and oxidants to various degrees. C. spectabilis proteases are differentially expressed in distinctive organs, and their stability against heat and oxidants makes this plant an important source of stable proteases.

Purification and subcellular localization of a secreted 75 kDa Trypanosoma cruzi serine oligopeptidase.

An extracellular serine peptidase was purified 460-fold from Trypanosoma cruzi epimastigotes culture supernatant with (NH(4))(2)SO(4) precipitation followed by affinity chromatography aprotinin-agarose and continuous elution electrophoresis, yielding a total recovery of 65%. The molecular mass of the active enzyme estimated by reducing and non-reducing SDS-PAGE was about 75kDa. The optimal pH and temperature of this glycosylated peptidase were 8.0 and 37 degrees C using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. The enzyme did not hydrolyze polypeptide substrates but was active against short peptide substrates containing arginine at the P1 site, in both ester and amide bonds. The peptidase was inhibited by TPCK and TCLK but not by other protease inhibitors suggesting that the enzyme belongs to the serine peptidase class. Interestingly, the enzyme seems to demonstrate some metal dependence since its activity was reduced by 1,10-phenanthroline, calcium and zinc ions. Rabbit anti-T. cruzi extracellular serine peptidase antiserum was used to show that the enzyme was restricted to intracellular structures, including the flagellar pocket, plasma membrane and cytoplasmic vesicles resembling reservosomes. These results suggest that the serine oligopeptidase is secreted into the extracellular environment through the flagellar pocket and the intracellular location could suggest its participation in certain proteolysis events in reservosomes. These findings show that this peptidase is a novel T. cruzi serine oligopeptidase, which differs not only from other peptidases described in the same parasite but also in other species of Trypanosoma.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.