The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.
Methods utilized frequently for the extraction of globulins from legume
seeds lead to a considerable
loss of these proteins in the albumin fraction due to a partial
solubilization of globulins in the albumin
extraction medium. The standard procedure to isolate legume seed
globulins was modified by (i)
including calcium and magnesium in the albumin extraction solution,
which completely solubilizes
the albumins free of contaminating globulins, and (ii) adding EDTA and
EGTA to the globulin
extraction solution, which efficiently extract the globulins.
Using this modified methodology, Lupinus
albus globulins were isolated and subsequently purified, and the
main individual globulins, namely
α-, β-, and γ-conglutins, were characterized. α- and
β-conglutins have been routinely considered as
the 11S (or legumin-like) and 7S (or vicilin-like), respectively,
protein components of L. albus seeds.
Using the total globulin fraction or the purified conglutins on
isopycnic sucrose or glycerol density
gradient centrifugation performed under low or high ionic strength and
on gel filtration techniques,
it is proposed that β-conglutin (with a sedimentation coefficient of
approximately 11 S) is considerably
heavier than α-conglutin (with a sedimentation coefficient of
approximately 7 S).
Keywords: Extraction; globulins; legume seeds; Lupinus albus; sedimentation
coefficients
The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.
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