Laura María Isabel López

Isolation and Characterization of a Cysteine Protease from the Latex of Araujia hortorum Fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 μM. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 μg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.

Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae)

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5–9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.

Laser microperforated biodegradable microbial polyhydroxyalkanoate substrates for tissue repair strategies: an infrared microspectroscopy study

AbstractFlexible and biodegradable film substrates prepared by solvent casting from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) solutions in chloroform were microperforated by ultraviolet laser ablation and subsequently characterized using infrared (IR) microspectroscopy and imaging techniques and scanning electron microscopy (SEM). Both transmission synchrotron IR microspectroscopy and attenuated total reflectance microspectroscopy measurements demonstrate variations in the polymer at the ablated pore rims, including evidence for changes in chemical structure and crystallinity. SEM results on microperforated PHBHV substrates after cell culture demonstrated that the physical and chemical changes observed in the biomaterial did not hinder cell migration through the pores. FigureComposition showing visible and IR images of a microperforated PHBHV film, with IR spectra showing crystallinity differences between bulk film and pore rim, schematic of cell growth and propagation strategy and SEM image showing evidence of cell growth on the underside of the biodegradable substrate.

Comparison of two cysteine endopeptidases from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

Abstract The properties of two cysteine peptidases (macrodontain I and II) isolated from fruits of Pseudananas macrodontes have been compared. The enzymes showed optimum pH ranges near neutrality and were inhibited by E-64 and other cysteine peptidase inhibitors. Molecular masses were 23459 and 23703 kDa, the isoelectric points were 6.1 and 5.9, and the K values were 13.4 and 8.9 M (BzPheValArg AMC) for macrodontain I and II, respectively. N? CBZLamino acid pnitrophenyl esters were tested for both enzymes. The Nterminal sequences of both proteases differed slightly and showed high sequence similarity to other pineapple stemderived cysteine endopeptidases.

Properties of a milk clotting protease isolated from fruits of Bromelia balansae Mez.

Abstract Unripe fruit extracts of Bromelia balansae Mez Bromeliaceae), whose principal endopeptidase is balansain I (isolated for anion exchange chromatography: pI = 5.45, molecular weight = 23192), exhibit pH profile with a maximum activity around pH 9.0 and are inhibited only by cysteine peptidases inhibitors. The alanine and glutamine derivatives of N?carbobenzoxy Lamino acid pnitrophenyl esters were strongly preferred by the enzyme. Enzymatic hydrolysis of milk and soy proteins yield characteristic patterns at pH 9.0. The Nterminal sequence showed very high homology (85 90%) with other known Bromeliaceae endopeptidases.

Surface modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer films for promoting interaction with bladder urothelial cells†‡

Often bladder dysfunction and diseases lead to therapeutic interventions that require partial or complete replacement of damaged tissue. For this reason, the development of biomaterials to repair the bladder by promoting the adhesion and growth of urothelial cells is of interest. With this aim, a modified copolyester of biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] was used as scaffold for porcine urothelial cell culture. In addition to good biocompatibility, the surface of P(HB-co-HV) substrates was modified to provide both, higher hydrophilicity and a better interaction with urothelial cells. Chemical treatments with ethylenediamine (ED) and sodium hydroxide (NaOH) led to substrate surfaces with decreasing hydrophobicity and provided functional groups that enable the grafting of bioactive molecules, such as a laminin derived YIGSR sequence. Physico-chemical properties of modified substrates were studied and compared with those of the pristine P(HB-co-HV). Urothelial cell morphology on treated substrates was studied. The results showed that focal attachment and cell-related properties were improved for peptide grafted polymer compared with both, the unmodified and functionalized copolyester.

Proteolytic activity in some Patagonian plants from Argentina

Six Patagonian plants were screened for proteolytic activity: Colliguaja integerrima, Euphorbia collina, E. peplus and Stillingia patagonica (Euphorbiaceae), Philibertia gilliesii (Asclepiadaceae) and Grindelia chiloensis (Asteraceae). P. gilliesii extracts showed the highest specific activity, followed by S. patagonica and E. collina. Proteolytic activity was unnoticeable in the other three species studied. Inhibition assays revealed that P. gilliesii and S. patagonica extracts contain cysteine-type peptidases and that in E. collina serine-type peptidases are present.

The surface modification of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers to improve the attachment of urothelial cells.

Biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] substrates were modified to improve the attachment of porcine urothelial cell culture. The pristine copolymer exhibits excellent mechanical properties to replace the bladder tissue, but its surface lacks chemical functionalities to interact with cells. Thus, wet chemical treatments based on NaOH and ethylenediamine in aqueous [ED(aq)] and isopropanol [ED(isoOH)] media to functionalize the P(HB-co-HHx) films surfaces were compared. Among these treatments, short ED(aq) treatment was able to decrease the hydrophobicity, rendering a surface with amino groups and without a significant alteration of the mechanical properties. Furthermore, to enhance the interaction with urothelial cells, laminin derived YIGSR sequence was covalently bound to these amino functionalized substrates. The focal attachment was clearly improved with this last treatment, comparing with those results found with the unmodified and first-step functionalized P(HB-co-HHx).

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (“oak leaved papaya”) latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman’s degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.