Olga Lima Tavares Machado

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties.

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.

Antimicrobial peptides and immunolocalization of a LTPin Vigna unguiculata seeds

Abstract Two cysteine-rich antimicrobial peptides (6.8 and 10xa0kDa) were isolated from cowpea ( Vigna unguiculata ) seeds and shown to deter development, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and F. solani and the yeast Saccharomyces cerevisiae . The peptides purified by RP-HPLC were submitted to automated N-terminal amino acid sequencing. Sequence analysis of these peptides showed the presence of a defensin and a lipid transfer protein (nsLTP) with high degree of homology to other antifungal peptides isolated from plants. LTP was detected in both cotyledon and embryonic axes of the seeds. Immunofluorescence assays also indicated that LTP was localized in the cell wall and in cytosolic compartments. In addition the presence of nsLTP was detected in seeds of different cultivars of cowpea and in three other leguminous seeds ( Vigna vexillata , Canavalia ensiformis and Phaseolus vulgaris ).

Purification and Characterization of a New Trypsin Inhibitor from Dimorphandra mollis Seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10−9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.

Isolation and partial characterization of a novel lectin from Talisia esculenta seeds that interferes with fungal growth

A novel plant lectin has been isolated from the seeds of Talisia esculenta and partially characterized. The purified lectin showed two protein bands in SDS-PAGE (20,000 and 40,000 kDa) and agglutinated human and animal erythrocytes. Of the various sugars tested, the lectin was best inhibited by mannose. A search of sequence databases showed that the N-terminal sequence had no homology to any known protein. The lectin inhibited the growth of the fungi Fusarium oxysporum, Colletotrichum lindemuthianum and Saccharomyces cerevisiae.

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata.

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.

Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds.

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.

The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus)

The seeds of the Jack bean, Canavalia ensiformis (L) DC are known to contain several toxic substances that prevent their utilisation as food for humans and animals. The lectin concanavalin A and the enzyme urease are the best known of these proteins. We have found that many proteins present in the seeds of the Jack bean, like trypsin inhibitors and canatoxin, are detrimental to the development of the bruchid insect Callosobruchus maculatus (F) (Coleoptera: Bruchidae). Among these proteins, canavalin (vicilin, 7S globulin) was found to be expressed in the seed coat. We suggest that seed coat canavalin, in addition to other detrimental proteins expressed in this tissue, may have been of importance in the evolutionary discrimination of the seeds of this legume by non‐pest bruchids.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata (pata-de-vaca, mororó), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants

Transgenic tomato plants (Lycopersicon esculentum) overexpressing the prosystemin transgene have been shown previously to accumulate a soluble 87 kDa cystatin constitutively. We report here that this protein can be found in a crystalline form which can be purified using a glycerol/sucrose gradient. Midgut homogenate of third-instar larvae of two coleopteran pest insects, Callosobruchus maculatus and Zabrotes subfasciatus, had their proteolytic activity content significantly inhibited by tomato cystatin (TC). In leaves of wild-type tomato plants, cystatin mRNA accumulated systemically in response to wounding, treatment with methyl jasmonate (MJ) and when supplied with systemin, corroborating the anti-herbivorous activity. Accumulation of cystatin mRNA occurred when plants were supplied with chitosan and oligogalacturonic acid fragments (OGA), suggesting an effect of TC against pathogens. Moreover, this protein reduced the growth of two fungi, Fusarium solani and Trichoderma viride in vitro. Taken together, the data reinforce a role for TC in defense response against pests or pathogens.

Plant insulin or glucokinin: a conflicting issue

The presence of insulin in plants is not accepted by the scientific community in general. In this review we discuss this paradigmand retrieve information that strongly suggests that insulin is indeed found in plants. We present results, which indicate that aprotein molecule with the same amino acid sequence as bovine insulin is expressed in leguminous plants. Additionally, weprovide evidence that proteins associated with insulin signalling pathways in vertebrates are also found in association withinsulin-like molecules in plants.