The purification and characteristics of purified HL60 tuftsin receptors are described. Purification was accomplished by affinity chromatography similar to that described earlier, wherein a tuftsin analog Thr-LysPro-Pro-Arg, is covalently linked at the Nα group to a solid support. The receptor consists presumably of two subunits approximately 66 KDa and 57 KDa. The dissociation constant of the receptor complex is 4.7 × 10−8 M with 5 × 104 receptors per cell. It can form oligomers with an Mr of about 560 KDa suggesting an octomeric structure, assuming the same number of each subunit is associated.

HL60 cells, rabbit peritoneal granulocytes or membrane preparations of these cells incorporate radioactivity when reacted with the radioactive peptide tuftsin [3H Pro3]-Thr-Lys-Pro-Arg. The radioactivity which is not diminished by repeated treatments with TCA and NaOH, is covalently bound to a membrane acceptor protein of 100 kDa. The peptide is not displaced by large concentrations of its constituent amino acids. The acceptor protein is resolved into one labeled peak by gel filtration on Sephadex G-200, Sephacryl S-300 and by SDS-PAGE. Digestion by trypsin and chymotrypsin results in the production of smaller fragments.

Many functions of monocyte/macrophage and granulocyte are activated by tuftsin; principally phagocytosis, motility, immunogenic stimulation, antibacterial and antineoplastic activities. Here it is shown that tuftsin stimulates HL60 growth to twice the control rate. The uptake of [ 3 H]uridine and [ 3 H]leucine in a pulse of 30 min was also double that of the control. The uptake of thymidine was not stimulated.

Tuftsin (Thr-Lys-Pro-Arg) receptor was purified to apparent homogeneity by affinity chromatography, using a pentapeptide analog (Thr-Lys-Pro-Pro-Arg) that binds the receptor more than 4 times as avidly as tuftsin. The analog was covalently linked to a solid support (Affi-Gel 10). Rabbit peritoneal granulocyte membrane solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was applied to the affinity column, the column was washed with 0.1 M ammonium carbonate (pH 7.9) and 0.1 M ammonium acetate (pH 5), and bound material was eluted with 20 nM tuftsin or pentapeptide. The eluate was concentrated and subjected to gel filtration; this yielded one major peak of [3H]tuftsin binding activity corresponding to approximately 500 kDa and a minor peak at approximately 250 kDa. Rechromatography of either peak resulted in the appearance of the same major and minor peaks. NaDodSO4/PAGE of the affinity-purified material under nonreducing conditions showed only two silver-staining bands. Electroblotting followed by [3H]tuftsin overlay and fluorography showed two adjacent radioactive bands corresponding in mobility to the silver-stained bands. Under reducing conditions, NaDodSO4/PAGE yielded molecular mass values 62 kDa and 52 kDa for the two tuftsin receptor subunits. Electron microscopy revealed a homogeneous population of spherical molecules with diameters of 104 A.

Tuftsin receptor was purified from rabbit peritoneal granulocytes by affinity chromatography. The pentapeptide analog, Thr-Lys-Pro-Pro-Arg was covalently linked to a solid support column. Rabbit granulocyte membrane was prepared, dissolved in 8 mM CHAPS and run through the column, eluted with 20 eta M free pentapeptide and subjected to dialysis concentration. When this was run on SDS-PAGE, two bands were obtained at a migration equivalent to Mr 60 and 62 K. These were electroblotted on nitrocellulose paper which showed two corresponding (/sup 3/H)-tuftsin binding bands. After reduction, and boiling, SDS-PAGE runs showed two bands Mr 85 and 70 K. When the purified receptor was reduced, alkylated and treated with endo-..beta..-N-acetylglucosaminidase H, only one band was obtained at Mr of about 90 K.

Tuftsin, Thr-Lys-Pro-Arg, that activates macrophage functions, binds to specific receptors on these cells. The receptor capacity to bind tuftsin is diminished by prior treatment of the cells with dithiothreitol. Adherent mouse peritoneal macrophages bind tuftsin to a far less extent than non-adherent macrophages. Michaelis constant (Km) of tuftsin for phagocytic stimulation of macrophages is 111 ηM. The half maximal binding concentration of tuftsin by these cells is 117 ηM. These are similar values and indicate that full occupancy of the receptors by tuftsin is a necessary prerequisite for maximal phagocytosis.

The characteristics of binding of tuftsin (Thr-Lys-Pro-Arg) and several of its synthetic analogues, to specific membrane receptors on rabbit polymorphonuclear granulocytes, were compared with the binding characteristics of rabbit specific antibody to tuftsin. [3H-Arg4]-tuftsin was used as the principal ligand. Six analogues were studied. Two of these, Thr-Lys-Pro and Ala-Lys-tuftsin-Glu-Ala3, showed no binding affinity either to receptor or antibody. Ala-Lys-tuftsin showed less binding than tuftsin to both acceptors. Three showed stronger binding than tuftsin. The order of binding among these was tuftsin ( [Glu]2-tuftsin ( [Pro-Pro3]-tuftsin (tuftsinyltuftsin. This same order of binding was found with both receptor and antibody.