Análisis fitoquímico y aplicación quimiotaxonómica de los mucílagos de origen foliar de las especies de Chorisia H.B.K. (Bombacaceae) que crecen en el país

El objetivo de este trabajo es el análisis de los mucílagos de hojas de las cuatro especies del género Chorisia H.B.K. que crecen en el país: Ch. insignis H.B.K., Ch. pubiflora (St.Hil) Dawson, Ch. speciosa St.Hil y Ch. crispiflora H.B.K. El estudio de las características físicas y químicas de los mismos, independientemente del aporte al conocimiento fitoquímico de especies de nuestra Flora, brinda la información básica ne cesaria para decidir su posible aprovechamiento industrial. Por otra parte existe la pretensión de utilizar los datos provistos por el análisis de estos mucílagos con fines quimiotaxonómicos , tanto a nivel genérico como específico.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Proteolytic properties of <i>Funastrum clausum</i> latex

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-a-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.Centro de Investigación de Proteínas Vegetale

Proteolytic properties of <i>Funastrum clausum</i> latex

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-a-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.Centro de Investigación de Proteínas Vegetale

Hydrolysis of caprine and ovine milk proteins, brought about by aspartic peptidases from Silybum marianum flowers

The flowers of cardoon (Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity – and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine as1- and b-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa – i.e. a fragment of b-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine as- and b-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in as1-casei

Proteolytic properties of <i>Funastrum clausum</i> latex

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-a-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.Centro de Investigación de Proteínas Vegetale

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain cII) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain cII was the minor proteolytic component in the latex, but showed higher specific activity than asclepain cI, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain cII displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain cII is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The N-terminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-l-Gln-p-nitrophenyl ester as substrate: Km = 0.1634 mM, kcat = 121.48 s-1, and kcat/Km = 7.4 × 105 s-1/mM.Centro de Investigación de Proteínas VegetalesFacultad de Ciencias Exacta

Sodium tetrathionate effect on papain purification from different <i>Carica papaya</i> latex crude extracts

Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption= ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.Centro de Investigación de Proteínas Vegetale

Sodium tetrathionate effect on papain purification from different <i>Carica papaya</i> latex crude extracts

Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption= ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.Centro de Investigación de Proteínas Vegetale

Sodium tetrathionate effect on papain purification from different <i>Carica papaya</i> latex crude extracts

Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption= ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.Centro de Investigación de Proteínas Vegetale

Proteasas de Bromeliaceae: V. Separación y purificación de sulfhidril - proteasas presentes en frutos de Bromelia balaasae Mez

Almost ripe fruits of Bromelia balansae Mez (Bromeliaceae) were blade homogeneized within cold (-20 O C ) acetone, providing a crude preparation that could be partialiy solubllized in phosphate buffer (pH 6,4) containing EDTA and cysteine 5 mM. Solutions hydrolize casein (maximum a c t ~ t ypH 6.25-8.5O), the activity being enhanced by addition of cysteine. The crude enzyme was purified by molecular sieve chromatography (Sephadex G-75, Superfme) and ion exchange chromatography (DEAE- and CM-Sepharose). Three proteolitically active fractions were thus obtainedLa trituración de frutos semimaduros de Bromelia balansae Mez (Bromeliaceae) en presencia de acetona fría (-20 OC) permite obtener una preparación enzimática cruda, que al ser tratada con buffer fosfatos de pH 6,4 conteniendo EDTA y cisteína 5 mM produce una solución con actividad proteolítica apreciable y con máxima acción hidrolítica sobre caseína dentro de un rango de pH entre 6,25 y 8,50. Esta solución enzimática cruda se purificó por cromatografía de exclusión molecular (Sephadex G-75 Superfuio) y de intercambio iónico (DEAE- y CM-Sepharosa), con lo que se obtuvieron tres fracciones proteolíticamente activas.El presente trabajo recibió el apoyo de la CIC y fue presentado en el X Congreso\nFarmacéutico Argentino (San Luis, 1988)