JBC, Vol. 252, Issue 9, 2967-2971, May, 1977

Recombinations of subunits of Phaseolus vulgaris isolectins

R. L. Felsted, M. J. Egorin, R. D. Leavitt and N. R. Bachur

Phaseolus vulgaris phytohemagglutinin is formed in vivo by the combination of erythrocyte (E)-reactive and lymphocyte (L)-reactive subunits into five tetrameric isolectins:L4,L3E1, L2E2, L1E3, and E4. Evidence for phytohemagglutinin subunit structure is obtained by in vitro dissociation of native isolectins in 6 M guanidine HCl followed by removal of dissociating agents to allow subunit recombination. Dissociation and recombination of L4 yielded a single protein, electrophoretically indistinguishable from the native L4. Similar treatment of E4 also yielded a single protein indistinguishable from native E4. Treatment of L3E1, L2E2, L1E3, or a mixture of L4 and E4, yielded five distinct proteins electrophoretically similar to all five native phytohemagglutinin isolectins. Milligram quantities of all five recombinant isolectins were prepared either from L2E2 or a mixture of L4 and L1E3 proportioned to yield equimolar quantitives of the two subunits on dissociation. The recombinant isolectins were purified by affinity and SP-Sephadex ion exchange chromatography. Electrophoretic and chromatographic properties and the erythroagglutinating and mitogenic activities of recombinant isolectins were essentially identical with the native isolectins. The inclusion of 125I-labeled L4 in the dissociation results in a distribution of 125I-labeled L subunit among the purified recombinant isolectins proportional to their proposed subunit structures.
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