Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.
Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.
Loetscher
H
H
Central Research Units, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Schlaeger
E J
EJ
Lahm
H W
HW
Pan
Y C
YC
Lesslauer
W
W
Brockhaus
M
M
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D014357:Trypsin; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D014357:Trypsin