Crystallization and preliminary studies of lima bean trypsin inhibitor

Crystals of lima bean trypsin inhibitor (LBTI) were obtained by using the vapor phase equilibration technique with sodium/potassium tartrate as the precipitating agent. The space group was determined to be cubic, I2₁3 with a= 110.2 Å. These crystals diffract to about 1.9 Å resolution. Preliminary analysis of self-rotation maps (calculated from native x-ray intensity data) suggests the presence of two monomers in the asymmetric unit. LBTI is very thermostable and retains activity even after boiling for 10 minutes. This property is exploited as part of its purification procedure. © 1997 Wiley-Liss, Inc.     

Cell binding and mitogenic properties of the lima bean lectins

Two lectins, a tetramer designated LBL₄ and an octamer LBL₈ designated have been purified from the lima beanPhaseolus lunatus. The tetramer appears to be nonmitogenic for human lymphocytes and is a weak mitogen for bovine cells. The octamer and a chemically cross-linked form of the tetramer are good mitogens. The lima bean lectin binds to only certain sub-populations of human lymphocytes. The primary class which does not bind appears to be a sub-population ofT-lymphocytes. Comparisons of cell binding with other lectins which bind to 2-acetamido-2-deoxy-D-galactose have been carried out. Quantitative analysis of the binding to human erythrocytes is co-operative but binding to lymphocytes is non-co-operative. These results show that there may not be a direct correlation between mitogenic stimulation and cooperative binding to membrane receptors.     

Inducible isoflavonoids from the lima bean,Phaseolus lunatus

Phaseolus lunatus seedlings treated with aqueous cuprous chloride produced 25 isoflavonoids which were isolated and characterized by UV, mass and ¹H NMR spectroscopy as kievitone, isoferreirin, 5-deoxykievitone, kievitone hydrate, cyclokievitone, cyclokievitone hydrate, daidzein, 2′-hydroxydaidzein, genistein, 2′-hydroxygenistein, 2,3-dehydrokievitone, luteone, phaseollidin, 2-(γ,γ-dimethylallyl)-phaseollidin, coumestrol, psoralidin, 7,8,2′,4′-tetrahydroxyisoflavone, 5,7,8,2′,4′-penta hydroxyisoflavone, 2,3-dehydrokievitol, lunatone, 5-deoxykievitol, kievitol, 3′-(γ,γ-dimethylallyl)-kievitone, 4-(γ,γ-dimethylallyl)-phaseollidin and 2-(γ,γ)-dimethylallyl)-6a-hydroxy-phaseollidin. The last nine compounds have novel structures. Possible biogenetic routes for these isoflavonoids and the taxonomic implications of their occurrence are discussed.     

Natural plant enzyme inhibitors: isolation and properties of a trypsin inhibitor from jack bean (Canavalia ensiformis)

A protease inhibitor which is equally active on bovine and porcine trypsins was isolated in a homogenous form from jack bean (Canavalia ensiformis). The preparation with a molecular weight of 18 kDa was found to be a glycoprotein with a high half cysteine content. Isoleucine and tyrosine were found to be absent. The inhibitor was heat-stable and stable at pH 2.0 and 11.0. It was ten times less active on bovine alpha-chymotrypsin and pronase than on trypsin. It displayed weak action on subtilisin BPN, porcine elastase and pepsin. The inhibitor was most effective in blocking the total proteolytic, tryptic and chymotryptic activities of rabbit pancreatic preparation. The relative ratios of inhibitions of the three activities on rabbit, bovine and human systems were respectively 1250:100:1, 600:100:1 and 46:18:1. While different substrates (except denatured serum albumin) did not significantly alter the magnitude of inhibition of bovine trypsin, the extent of inhibition of bovine alpha-chymotrypsin by the jack bean inhibitor was highly dependent on the substrate used in the assay.     

Sucrose metabolism in lima bean seeds

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.     

An additional gametophyte factor in the lima bean

Summary A second gametophyte factor (Ga ₂) is described in lima beans. This gene produces an interaction between the male gametophyte and style such that: (a)Ga ₂ pollen functions irrespective of stylar genotype; (b)ga ₂ pollen is functional onga/ga styles but is unable to compete withGa ₂ pollen onGa ₂/Ga ₂ orGa ₂/ga ₂ styles.Ga ₂/ga ₂ is linked with theP/p locus (recombination fraction =0.244±.017); it is therefore probably located in linkage group I.Some similarities and differences between gametophyte factors and incompatibility systems and some factors which might influence the establishment and maintenance ofga alleles in populations are discussed. Evidence is presented that theGa ₂ allele arose as a recent mutationga ₂Ga ₂ in one of the pure-line parents of the families studied.

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Zusammenfassung An Limabohnen-Familien, die für das GenP/p für Samenschalenfarbe heterozygot sind, wurden persistente Spaltungsabweichungen analysiert.Ein zweiter gametophytischer Faktor (Ga ₂) wird beschrieben. Dieses Gen bewirkt eine Interaktion zwischen dem männlichen Gametophyten und dem Griffel in der Form, daß a)Ga ₂-Pollen unabhängig von dem Genotyp des Griffels funktionsfähig ist. b)ga ₂-Pollen zwarga/ga-Griffel befruchten, aber aufGa ₂/Ga ₂-Griffeln oderGa ₂/ga ₂-Griffeln mitGa ₂-Pollen nicht konkurrieren kann.Ga ₂/ga ₂ ist mit dem LocusP/p gekoppelt (Rekombinationswert 0,244±0,017) und daher wahrscheinlich in Koppelungsgruppe I lokalisiert.Ähnlichkeiten und Unterschiede zwischen gametophytischen Faktoren und Inkompatibilitätssystemen sowie Faktoren, die das Auftreten und die Ausbreitung vonga-Allelen in Populationen beeinflussen können, werden diskutiert. Es wird der Beweis erbracht, daß dasGa ₂-Allel in einer der reinen Elternlinien der untersuchten Familien als eine neue Mutationga ₂Ga ₂ entstanden ist.

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This work was supported in part by a grant from the National Science Foundation G-14991.