José Xavier-Filho

Resolution and partial characterization of proteinases and α-amylases from midguts of larvae of the bruchid beetle Callosobruchus maculatus (F.)

Abstract 1. 1. Three proteinases and four α-amylases wre identified in Callosobruchus maculatus larvae. 2. 2. All proteinases have the same M r (25,000) and optimum pH between 5.5 and 6.0 and are inhibited by iodoacetic acid, iodoacetamide, pCMB, TLCK, TPCK and E-64. Protein inhibitors of cysteine proteinases isolated from Vigna unguiculata seeds and from chicken egg white also inhibited all three proteinases. 3. 3. The four α-amylases have optimum pH between 5.2 and 6.0 and mol. wt of 56,000, 45,000, 36,000 and 33,000. None of them are inhibited by an α-amylase inhibitor from Phaseolus vulgaris seeds. An α-amylase inhibitor from wheat inhibited the amylases of M r 45,000, 36,000 and 33,000, but failed to inhibit the α-amylase of M r 56,000. 4. 4. The possible role of proteinase and α-amylase inhibitors in proteing V. unguiculata against attack by C. maculatus is discussed.

Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus)

Abstract 1. A globulin fraction prepared from the meal of Callosobruchus maculatus -resistant cowpea ( Vigna wiguiculata ) seeds was shown to be detrimental to this bruchid when incorporated in artificial seeds. 2. The performance of C. maculatus was also shown to be strongly hindered by vicilins from resistant seeds when these storage proteins were incorporated in artificial seeds at the level of 2%. 3. The purified vicilins from seeds of both resistant and susceptible cowpea varieties were shown to have the same SDS-PAGE pattern but different mobilities in non-denaturing polyacrylamide gel electrophoresis. 4. These results and previous ones obtained by us (Silva and Xavier-Filho, 1991; Sales et al. , 1992) strongly suggest that the resistance of cowpea seeds from the cultivar TVu 2027 and from others bred from it is associated with the presence of vicilin molecules which are refractory to digestion by bruchid midgut proteinases.

The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.

Proteinases and amylases of larval midgut of Zabrotes subfasciatus reared on cowpea (Vigna unguiculata) seeds.

Proteinase and amylase activities in larval midguts of the bruchid beetle Zabrotes subfasciatus (Boh.) (Coleoptera: Bruchidae) reared on cowpea (Vigna unguiculata (L.) Walp.) seeds were investigated. We could detect and isolate a proteolytic activity with a pH optimum of 5.5 (on azo‐casein as substrate) which was activated by thiol reagents and inhibited by several compounds reactive against ‐SH groups. None of the plant protein inhibitors of serine proteinases utilized were effective inhibitors of this activity. This activity has characteristics of a cysteine class proteinase. We could also detect and isolate a proteolytic activity with a pH optimum of 3.5 (on hemoglobin as substrate) which was not influenced by activators or inhibitors of cysteine, serine, or metalloproteinases. This activity was totally inhibited by pepstatin, a specific inhibitor of aspartic proteinases. We conclude that this activity is due to an aspartic class proteinase. We found also that the aspartic class proteolytic activity is higher than the cysteine class proteinase activity in the midguts of Z. subfasciatus. This seems to be contrary to what is found in Callosobruchus maculatus (F.) larvae midguts. An amylolytic activity with the charateristics of an α‐amylase was also detected and isolated.

Comparison between the levels of aspartic and cysteine proteinases of the larval midguts of Callosobruchus maculatus (F.) and Zabrotes subfasciatus (Boh.) (coleoptera : bruchidae)

Abstract 1. 1. An acidic proteolytic enzyme acting on hemoglobin at optimum pH 3.3 was partially purified from Callosobruchus maculatus larval midgut. 2. 2. The proteinase showed a mol. wt of 62,000 and was inhibited by pepstatin and diazoacetyl norleucyl methyl ester, which are known inhibitors of aspartic proteinases. 3. 3. Both aspartic and cysteine proteinases were measured in larval midgut homogenates of C. maculatus and Zabrotes subfasciatus. 4. 4. The higher relative amounts of these enzymes in larval midgut of Z. subfasciatus suggest that this bruchid has a higher capacity to digest proteins of some cowpea seeds than C. maculatus has.

The amino acid sequence and reactive (inhibitory) site of the major trypsin isoinhibitor (DE5) isolated from seeds of the Brazilian Carolina tree (Adenanthera pavonina L.)

Abstract Eight iso-inhibitors of trypsin were isolated from seeds of the Brazilian Carolina tree ( Adenanthera pavonina L.) by precipitation with ammonium sulphate, gel filtration, affinity chromatography on enzymatically inert anhydrotrypsin Sepharose 4B, and separated by ion-exchange chromatography on DEAE-Sepharose. The p I values of the isoinhibitors (DE1-DE8) ranged from 5.10 to 4.40. Each isoinhibitor had an M r of approx. 21 000 and was composed of a large α chain ( M r 16 000) and a smaller β chain ( M r 5000) linked together by a disulphide bond. The complete amino acid sequence of isoinhibitor DE5 (p I 4.75) was deduced by analysis of peptides and fragments derived from the separated α and β chains by digestion with trypsin, chymotrypsin, pepsin, thermolysin, the Staphylococcus aureus V8 proteinase and iodosobenzoic acid. The sequence of the Carolina DE5 isoinhibitor and the location of its reactive (trypsin-inhibitory) peptide bond showed clear homology with the Kunitz-type proteinase inhibitors from soybean, winged bean and a number of other legume seeds.

Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on α-amylases and oligosaccharidases

Abstract Determinations of carbohydrases, aminopeptidases and acid phosphatase in the larval midgut cells and in the luminal contents of Callosobruchus maculatus and Zabrotes subfasciatus have been carried out. The results showed that larvae of both species displayed similar distribution of digestive enzymes in the intestinal compartments. Most larval digestive enzyme activities were found in the luminal contents. Of the activities found in the midgut tissue, only aminopeptidase is predominant in the membrane fraction. Comparisons of activities recovered from a seed flour mass equivalent to the midgut mass showed that a high percentage of the luminal aminopeptidase activity and, to a lesser extent, α-galactosidase activity, can be derived from the seeds, whereas the other enzymes are produced by the insects. Activities against starch, maltose and maltodextrins were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on differences in elution profiles on hydrophobic chromatography and banding patterns in mildly denaturing electrophoresis, both species showed a multiplicity of glycosidases. The data suggest that the majority of carbohydrate digestion occurs in the midgut lumen, whereas protein digestion should take place partly in the lumen and partly at the cell surface. Larvae of Z. subfasciatus can modulate the levels of α-amylases and α-glucosidase in response to different diets. The complex of carbohydrases found is qualitatively appropriate to digest the free oligosaccharides and oligomaltodextrins produced by α-amylases from the starch granules of host seeds.

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insects midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.

Legume seed vicilins (7S storage proteins) interfere with the development of the cowpea weevil (Callosobruchus maculatus (F)).

Vicilins (7S storage proteins) isolated from the seeds of the legumes Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), Canavalia ensiformis (jack bean), Glycine max (soybean), Phaseolus vulgaris (common bean) and Phaseolus lunatus (lima bean) were shown to be immunologically related and to bind to a chitin matrix. The effect of the isolated vicilins on the development of the cowpea weevil Callosobruchus maculatus was examined. Vicilins from all non-host seeds, including those of the C maculatus-resistant cowpea line IT81D-1045, strongly inhibited larval development (ED 50 of 1.07 ± 0.32% to 2.22 ± 0.64%). Vicilins from the C maculatus-susceptible cowpea CE-31 and adzuki bean seeds were the exception with ED 50 of 6.25 ± 0.75% and 5.40 ± 1.54%, respectively. These results correlate well with the host range of C maculatus and are possibly a reflection of the low digestibility of vicilins by insect midgut proteinases in addition to the ability they show in binding to chitin-containing structures like the ones found in the bruchid midguts.

Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells.

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea (Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H+ pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.