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

Campos, Francisco; Xavier‐Filho, J.; Silva, Carlos P.; Ary, Maria B.

doi:10.1016/0305-0491(89)90312-x

Cited by 73 publications

(59 citation statements)

References 11 publications

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“…We determined the concentrations of PHA-P and purified aAI needed to cause 50% inhibition of porcine a-amylase or cowpea weevil a-amylase in vitro (3). The results show that porcine a-amylase is inhibited 50% at 10.2 ± 1.5 Ag/mL of purified common bean aAI.…”

Section: And Discussionmentioning

confidence: 99%

“…The bean aAI was purified as described (16). Assay of a-amylase activity was similar to that previously described (3,7). Assay of porcine a-amylase was performed by suitably diluting in reaction buffer the enzyme to give, in the absence ofinhibitor, hydrolysis kinetics in the linear range.…”

Section: Assays Of Lectins and Lectin-like Proteinsmentioning

confidence: 99%

“…Cowpea weevil midgut preparations containing aamylase activity were suitably diluted and assayed as described above (3,7). RESULTS …”

Section: Insect Midgut Assaysmentioning

confidence: 99%

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α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

Huesing

Shade²,

Chrispeels³

et al. 1991

Plant Physiol.

109

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There are claims that phytohemagglutinin (PHA), the lectin of common bean, Phaseolus vulgaris, is toxic when fed to the cowpea weevil, Callosobruchus maculatus, and that PHA serves as the chemical defense against this seed-feeding bruchid beetle (DH Janzen, HB Juster, IE Liener [1976] (1,4,8,9), one role was proposed by Janzen et al. (12), who showed that 1% (w/w) Phaseolus vulgaris lectin (PHA) from black bean caused mortality when incorporated into the diet of the cowpea weevil, Callosobruchus maculatus F. (Coleoptera: Bruchidae). They concluded that "a major part ofthe adaptive significance of phytohemagglutinins (PHA) in black bean and other legume seeds is to protect them from attack by insect seed predators" (12). Subsequently, the PHA-mediated cowpea/ common bean interaction has become a prime example of protein-mediated plant-insect interactions.Gatehouse et al. (6) (also P. vulgaris) was toxic to the cowpea weevil. Curiously, they noted that a highly purified preparation (>95%) of PHA was less toxic than a less pure commercial preparation obtained from Sigma Chemical Co., London (6). PHA is a tetrameric protein composed of two different subunit types: one erythrocyte-binding (E-type) and one lymphocyte-binding (L-type) (9). Gatehouse et al. (6) concluded that the Etype subunit was the major antimetabolite. However, subsequent studies by Boulter (2) indicated that purified E-type or L-type isolectins were both ineffective when tested alone against the cowpea weevil. To account for the biological activity of the mixture described in the earlier paper (6), Boulter (2) concluded that there must be a synergistic effect between the two different lectin subunits.These provocative results and conclusions led us to determine ifplant lectins represent a general category ofinsecticidal proteins (18). We screened 17 different commercially available plant lectins in our artificial seed bioassay system (21) using the cowpea weevil (18). Included in the study were a PHA lectin mixture (PHA isolectins composed of both the E and L subunits), as well as purified PHA-E and PHA-L. Our results showed that none ofthe three PHA lectin preparations were effective against the cowpea weevil when fed at 1 % (w/ w) in the diet (18), a level claimed effective in the published studies (6,12). Since we had shown that other lectins show great promise as biotechnology based plant chemical defenses (18,22) and because PHA is being advanced as a potential candidate for this role by some investigators, we decided to investigate the putative toxic qualities of PHA further. MATERIALS AND METHODS Artificial SeedsTo test the effects ofvarious potential toxins on seed feeding insects, we used an artificial seed system previously developed in our laboratory (21). Briefly, the system involved milling decorticated cowpea seeds (cultivar California Blackeye No. 5) into flour, adding aqueous solutions of test compounds to the flour to make a paste, injecting the paste into Teflon molds, freezing the paste on dry ice and then liquid N2, ...

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Section: And Discussionmentioning

confidence: 99%

Section: Assays Of Lectins and Lectin-like Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

Huesing

Shade²,

Chrispeels³

et al. 1991

Plant Physiol.

109

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“…This cleavage removes a small C-terminal peptide and makes the inhibitor ineffective against pancreatic a-amylase. Cys proteases rather than Ser proteases are abundant in larval midguts of bruchids and may be primarily responsible for protein digestion (Kitch and Murdock, 1986;Campos et al, 1989). However, prevention of proteolysis by protease inhibitors shows that the midgut protease responsible for the cleavage of aAI-1 is a Ser protease.…”

Section: How Do Bruchids Tolerate High Levels Of Aai In Beans?mentioning

confidence: 99%

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

1996

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~The seeds of starchy grain legumes are an important staple in many countries, but as with other crops, insect pests can cause considerable crop losses. The postharvest damage done by larvae of bruchids (Co1eoptera:Bruchidae) can be quite extensive. Crops of the common bean (Pkaseolus vulgaris), the scarlet runner bean (Phaseoltis coccineus), and the tepary bean (Phaseolus acutifolius) can be severely damaged by the bean weevil (Acanthoscelides obtectus) and the Mexican bean weevil (Zabrotes subfasciatus) . This damage occurs ín spite of the presence in the seeds of a family of plant defense proteins that comprises PHA and aAI (see Chrispeels and Raikhel, 1991). The three Phaseolus species are not equally sensitive to the bruchids. For example, tepary bean is more resistant than common bean to the bean weevil (Shade et al., 1987).

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“…The effect of temperature on α-amylase activity was determined either by incubating the reaction mixture at 10,15,20,25,30,35,40,45, 50, 55, 60 and 70°C for 30 min. The effect of temperature on stability of amylase activity was tested by pre-incubation of the enzyme at 10, 20, 30, 40, 50, 60 and 70°C for 30 min, followed by measurement of activity as mentioned before.…”

Section: Effect Of Ph and Temperature On Enzyme Activitymentioning

confidence: 99%

Assessing of α-Amylase Activity of Midgut in Wheat Bug Eurygaster maura

Mehrabadi¹,

Bandani²

2009

American J. of Applied Sciences

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Abstract:To determine of α-amylase activity in the midgut of Eurygaster maura, some of adult insect was collected and their gut isolated and characterized. Enzyme samples from midguts of adults were prepared by the method of Cohen with slight modifications. α-Amylase activity was assayed based on Bernfeld method by the dinitrosalicylic acid (DNS) procedure. The activity of α-amylase in midgut was 0.083 U/insect. The optimum pH and temperature for the enzyme activity was determined to be 6-6.5 and 30-35ºC, respectively. The enzyme activity was inhibited by addition of EDTA (Ethylenediamine tetraacetic acid) urea, CaCl2, MgCl2 and SDS but Mg 2+ , NaCl and KCl enhanced enzyme activity.

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

Cited by 73 publications

References 11 publications

α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

α-Amylase Inhibitor, Not Phytohemagglutinin, Explains Resistance of Common Bean Seeds to Cowpea Weevil

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

Assessing of α-Amylase Activity of Midgut in Wheat Bug Eurygaster maura

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