Singer J A, Morrison M
Biochim Biophys Acta. 1975 Nov 3;406(4):553-63. doi: 10.1016/0005-2736(75)90032-2.
A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.
开发了一种可重复的定量测定方法，用于检测凝集素介导的人红细胞凝集，该方法基于凝集和未凝集红细胞沉降速率的差异。此测定法用于研究植物血凝素-P对人红细胞的凝集作用。发现植物血凝素-P对人红细胞的凝集作用取决于细胞的代谢状态。代谢耗尽的红细胞比供应腺苷的类似细胞更难凝集。这并非由于细胞肿胀和僵硬，因为低渗溶液中的红细胞在植物血凝素-P凝集方面未表现出显著变化。代谢耗尽的红细胞，或储存8周的血液中的红细胞，在ATP存在下裂解并重新封闭后，被植物血凝素-P凝集的程度比无ATP的对照样品大得多。单独的Mg2+或与ATP一起存在时，对重新封闭的膜的凝集性影响很小。低浓度的Ca2+（0.2 mM）对凝集性影响不大，尽管高浓度的Ca2+（5 mM）会在一定程度上抑制重新封闭的膜的凝集性。代谢耗尽的红细胞和先前用腺苷处理过的耗尽红细胞，在用胰蛋白酶处理后释放出相似量的唾液酸。用胰蛋白酶处理过的补充腺苷的细胞比用胰蛋白酶处理过的代谢耗尽细胞更容易凝集。红细胞的凝集不受细胞松弛素B（40微克/毫升）的影响。长春花碱（0.2 mM）使耗尽的红细胞凝集情况与补充腺苷的红细胞相似，但对补充腺苷的红细胞的凝集没有影响。得出的结论是，人红细胞中的ATP可能参与调节植物血凝素-P的凝集性。这不是红细胞中已知伴随ATP耗尽的更僵硬膜的结果，也不是ATP水平对Ca2+或Mg2+含量影响的结果。似乎ATP可能通过与膜相关成分直接或间接相互作用来调节人红细胞对植物血凝素-P的凝集性，该膜相关成分可能也对长春花碱敏感。
