Critical evaluation of proteomic protocols for passion fruit (Passiflora edulis Sims) leaves, a crop with juice market benefits

Passion fruit grows practically all over Brazilian territory; its production is largely destined to juice industry and expanding to overseas markets. The suitability of four protein extraction protocols for plant proteome was  investigated to determine the best choice for studies concerning passion fruit leaf proteins. Trichloroacetic acid (TCA)/acetone extraction; isoelectric  focusing (IEF) buffer extraction; phenol (Phe) extraction and Phe-SDS extraction were tested. The Phe method produced the best results, showing higher reproducibility of resolved protein spots and clearer 2D gel  background staining. In comparison, the Phe-SDS method presented fewer spots and lower reproducibility. The TCA/acetone method produced the fewest identifiable spots and the IEF buffer produced the poorest results,displaying fewer reproducibly detected spots, more vertical streaks and darker 2D staining. Selected spots, obtained with Phe method, were identified by spectrometric analysis (MALDI-TOF-TOF) to exemplify the viability to perform more comprehensive proteomic studies with passion fruit leaves and, therefore increase information about stress-related and developmental responses in this fruit crop.Key words: Passion fruit, proteomic, protein extraction, juice industry

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 K-i of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site. (C) 2000 Elsevier Science Ltd. All rights reserved.54655355

Isolation and characterization of isolectins from Talisia esculenta seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV ) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm x 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M-r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.20649550

Effect of a toxic protein isolated from Zea mays seeds on the development and survival of the cowpea weevil, Callosobruchus maculatus

A new toxic glycoprotein (Zeatoxin) has been isolated from Zea mays and sequenced. The toxin is a single polypeptide chain, with Mr of 10 kDa. Zeatoxin was not digested by a mixture of pepsin and papain or by midgut preparations of C. maculatus. A diet of artificial seeds (final concentration, 0.25%) was lethal to 50% of C. maculatus. These results show that Zeatoxin is a new toxic glycoprotein to C. maculatus. A search in the database indicated that the N-terminal sequence show no homology to any other known protein.7422523

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. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.401616

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 x 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.Univ Fed Mato Grosso do Sul, Dept Ciencias Nat Tres Lagoas, CEUL, BR-79603011 Tres Lagoas, MS, BrazilUniv Estadual Campinas, Dept Bioquim, Inst Biol, Campinas, SP, BrazilEscola Paulista Med, Dept Bioquim, BR-04023 São Paulo, BrazilUniv Estadual Norte Fluminense, Ctr Biociencias & Biotecnol, Campo Dos Goytacazes, RJ, BrazilEscola Paulista Med, Dept Bioquim, BR-04023 São Paulo, BrazilWeb of Scienc