The binding of phytohemagglutinin M to rat spleen lymphocytes. Quantitative studies.
Phytohemagglutinin M (PHAM) has been purified from the commercial mixture of proteins produced by Phaseolus vulgaris, using a Sepharose-thyroglobulin column. The protein gave one band on gel electrophoresis and two bands on SDS-gel electrophoresis (mol. wt. 33 700 and 32 100, respectively). Molecular weight determination by ultracentrifugation gave a value of 61 200 +/- 700. The protein had a minimum sugar content of 16%. Binding studies of PHAM to purified rat spleen lymphocytes have been performed at 0, 25, and 37 degrees C. It was shown that the cells bound about the same amount of lectin at 0 and 37 degrees C, but less protein was bound at 25 degrees C. The binding phenomenon showed saturability at all temperatures. Data were analyzed by Scatchard plots and two kinds of binding sites were found. High-affinity sites and low-affinity sites have been characterized in terms of association constants and (apparent) number. It was also shown that cells treated with trypsin or sodium azide bound less lectin. Bound concanavalin A did not appear to affect the amount of bound PHAM, but its influence was reflected in the value of the association constant for the binding of PHAM. Unlabelled PHAM was shown to displace radioactive PHAM from the cells, but could not remove bound concanavalin A. The significance of these results is discussed in terms of the fluid plasma membrane model and cellular metabolism.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D003208:Concanavalin A / Q000494:pharmacology; D007700:Kinetics; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D013154:Spleen / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D001386:Azides; D037102:Lectins; D003208:Concanavalin A; D014357:Trypsin