Isolation of GTP-binding proteins from myeloid HL-60 cells. Identification of two pertussis toxin substrates.
We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.
D037361:Adenylate Cyclase Toxin; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D008970:Molecular Weight; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D037361:Adenylate Cyclase Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins