A1 and A2 erythrocytes can be distinguished by reagents that do not detect structural differences between the two cell types

The differential reactivity of four mouse monoclonal antibodies (AbCB, AbHT29-36, AbM2, and AbS12) and Dolichos biflorus lectin with A1 and A2 erythrocytes was analyzed. Only AbS12 and D. biflorus lectin were able to preferentially agglutinate A1 erythrocytes. AbS12 is known to react only with short chain, unbranched structures (such as Aa-2 and Ab-2 glycolipids) and not with longer chains or with type 3 and type 4 structures. D. biflorus was shown to have a similar specificity by lectin staining of glycolipids separated by thin-layer chromatography. Analysis of the binding of radiolabeled AbCB and AbS12 to A1 and A2 erythrocytes by Scatchard analysis showed that, whereas the former antibody recognizes high-affinity sites on both A1 and A2 cells, AbS12 reacts with high-affinity sites only on A1 cells. Because A1 and A2 erythrocytes have a similar complement of short chain type 2 glycolipids, although in different amounts, it is suggested that AbS12 and D. biflorus lectin differentiate between the two cell types on the basis of quantitative, nonstructural features. This is in contrast to AbTH1, which reacts with a repetitive A epitope (type 3 A chain) and distinguishes between A1 and A2 cells based on the preferential expression of type 3 A chains in A1 erythrocytes. Thus, two views of A1/A2, i.e., qualitative vs quantitative are correct, depending on the properties of the reagent being used to distinguish between the two cell types.     

A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies

Human mitochondrial F(1)F(0) ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F(1) in a 96-well microplate activity assay system, to establish a method for fast high throughput screening of inhibitors, toxins, and drugs with very small amounts of enzyme. For preparative purification, mitochondria from human heart tissue as well as cultured fibroblasts were solubilized with dodecyl-beta-d-maltoside, and the F(1)F(0) was isolated with anti-F(1) monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F(1)F(0) contained a full complement of subunits that were identified with specific antibodies against five of the subunits (alpha, beta, OSCP, d, and IF(1)) and by MALDI-TOF and/or LC/MS/MS for all subunits except subunit c, which could not be resolved by these methods because of the limits of detection. Microscale immunocapture of F(1)F(0) from detergent-solubilized mitochondria or whole cell fibroblast extracts was performed using anti-F(1) monoclonal antibody immobilized on 96-well microplates. The captured complex V displayed ATP hydrolysis activity that was fully oligomycin and inhibitor protein IF(1)-sensitive. Moreover, IF(1) could be co-isolated with F(1)F(0) when the immunocapture procedure was carried out at pH 6.5 but was absent when the ATP synthase was isolated at pH 8.0. Immunocaptured F(1)F(0) lacking IF(1) could be inhibited by more than 90% by addition of recombinant inhibitor protein, and conversely, F(1)F(0) containing IF(1) could be activated more than 10-fold by brief exposure to pH 8.0, inducing the release of inhibitor protein. With this microplate system an ATP hydrolysis assay of complex V could be carried out with as little as 10 ng of heart mitochondria/well and as few as 3 x 10(4) cells/well from fibroblast cultures. The system is therefore suitable to screen patient-derived samples for alterations in amount or functionality of both the F(1)F(0) ATPase and IF(1).