Isolation and characterization of a trypsin inhibitor from lima beans

Fraenkel‐Conrat, H.; Bean, R. C.; Ducay, Eufemio D.; Olcott, Henry Steel

doi:10.1016/0003-9861(52)90200-2

Cited by 87 publications

(13 citation statements)

References 15 publications

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“…In respect of its high sulphur content and low molecular weight, hevein resembles the trypsininhibiting protein from lima beans reported by Fraenkel-Conrat, Bean, Ducay & Olcott (1952).…”

Section: Discussionsupporting

confidence: 51%

The proteins of Hevea brasiliensis latex. 4. Isolation and characterization of crystalline hevein

Archer

1960

Biochemical Journal

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Fresh rubber latex obtained from Hevea bra8iliensis consists of a suspension of several types of particles in an aqueous serum, and can be separated into fractions by high-speed centrifuging (Cook & Sekhar, 1953). The top layer in the centrifuge tube consists mainly of rubber particles, below which is a layer of almost clear serum. At the bottom of the tube is a layer referred to in this paper as the 'bottom fraction', which contains sedimentable bodies including the lutoid particles described by Homans & van Gils (1948). In preliminary work an aqueous extract of the bottom fraction was found to contain several electrophoretically distinct protein components. This observation has been confirmed and elaborated by Moir & Tata (1960). This paper describes the isolation and characterization of a major component which is anionic at pH 8-6 and for which the name hevein is proposed. EXPERIMENTAL Materials. Freeze-dried samples of the bottom fraction of freshly tapped latex were used throughout. Collection of the latex and centrifugal separation and freeze-drying of the bottom fraction were carried out by the staff of the Rubber Research Institute of Malaya, as described by McMullen (1959). All freeze-dried fractions were sealed under high vacuum in Malaya, and on arrival at our Laboratory were stored at-150 until required. Elementary analyses. Total N was determined by the micro-Kjeldahl method and C, H and ash by Pregl microcombustion. The S content of the ash and gaseous combustion products was measured turbidimetrically as BaSO4 after complete dissolution of the ash in dilute HC1. Where the amount of material available for analysis was limited, and the ash content was known to be low (as in purified hevein), C, H and S were determined on the same sample with a microcombustion tube containing a roll of silver and PbO2 only. P was determined colorimetrically with ammonium molybdate after wet oxidation. Soluble proteins were extracted from the freeze-dried solids with 0*05M-phosphate buffer, pH 6-8, or with 0-05 m-Na2B407, pH 9-3. The protein-N content of the extract was then determined by tannic acid precipitation and Kjeldahl analysis (Archer & Sekhar, 1955). Purified protein samples were dried to constant weight in vacuo at 800 before analysis.

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“…In respect of its high sulphur content and low molecular weight, hevein resembles the trypsininhibiting protein from lima beans reported by Fraenkel-Conrat, Bean, Ducay & Olcott (1952).…”

Section: Discussionsupporting

confidence: 51%

The proteins of Hevea brasiliensis latex. 4. Isolation and characterization of crystalline hevein

Archer

1960

Biochemical Journal

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“…The data presented here clearly demonstrate that mung bean cotyledons contain a trypsin inhibitor which resembles one of the two trypsin inhibitors found in black-eyed peas (7) but is unlike the trypsin inhibitor of soybeans (10) or lima beans (6). The mung bean trypsin inhibitor does not inhibit chymotrypsin and the antibodies against it do not react with soybean extracts.…”

Section: Resultsmentioning

confidence: 66%

“…It is therefore different from the soybean trypsin inhibitor described Kunitz (10) or the lima bean trypsin inhibitor characterized by Fraenkel-Conrat et at. (6). Gennis and Cantor (7) found two trypsin inhibitors in the related species Vigna unguiculata (black-eyed pea), one which only inhibits trypsin and one which inhibits chymotrypsin as well.…”

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confidence: 99%

Trypsin Inhibitor in Mung Bean Cotyledons

Chrispeels¹,

Baumgärtner²

1978

Plant Physiol.

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Trypsin inhibitor was purHied to homogeneity from seeds of the mung bean ( Vigna radiats ILl Wilczek). The protease inhibitor has the foUowing properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel ifitration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptdase in the cotyledon of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of,B-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts. Seed germination and the subsequent growth of the young seedlings are characterized by the breakdown of the reserve proteins of the seed. This breakdown is accompanied at an enhancement of protease-especially endopeptidase-activity, as well as by a decrease in protease inhibitor activity. Seeds of the Leguminosae, the Graminae, and the Solanaceae are an abundant source of protease inhibitors, and especially trypsin-or chymotrypsin-inhibitors, but the role of these inhibitors in the physiology of the plants is not understood. It has been proposed that these proteinaceous inhibitors may be storage proteins, that they play a role in masking the activity of preexisting plant proteases, and that they protect the seeds against pathogens and phytophagous insects (for reviews see Liener and Kakade (11) Shain and Mayer (20) reported that a trypsin inhibitor in lettuce seeds inhibits an endogenous trypsin-like enzyme which increases during germination, but the function of this enzyme in reserve protein breakdown is unknown. Inhibitors of endogenous plant proteases have been found in rice (9), barley (14), and in mung bean (1). However, several studies led to the conclusion that the great increase in endopeptidase in the storage tissues during seed germination cannot be accounted for by the inactivation of an "equivalent amount" of inhibitors (1, 14).Recently Miege and collaborators (13,17,18)

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“…In all cases the KPTI used was a crystalline, salt-free material (Worthington Biochemical Corp.). The SBTI was a three-times-crystallized preparation (Worthington Biochemical Corp.), and the LBTI (Worthington Biochemical Corp.) was fraction 111 of the preparation described by Fraenkel-Conrat et al (1952). The trypsin used was a two-timescrystallized preparation obtained from Worthington Biochemical Corp.…”

Section: Methodsmentioning

confidence: 99%