The G alpha z gene product in human erythrocytes. Identification as a 41-kilodalton protein.
A cDNA encoding a previously unknown G protein alpha-subunit lacking the site for pertussis toxin-catalyzed ADP-ribosylation was recently cloned and its putative protein product named Gz (Fong, H. K. W., Yoshimoto, K. K., Eversole-Cire, P., and Simon, M. I. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3066-3070) or Gx (Matsuoka, M., Itoh, H. Kozasa, T., and Kaziro, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5384-5388). A synthetic peptide corresponding to the deduced carboxyl-terminal decapeptide of this putative protein (alpha z) has been synthesized and used to prepare a polyclonal rabbit antiserum directed against the protein. The specificity and cross-reactivity of this antiserum was assessed using bacterially expressed recombinant G protein alpha-subunit fusion proteins (r alpha). The crude antiserum strongly recognizes r alpha z in immunoblots. Pretreatment of antiserum with antigen peptide greatly reduces the interaction of the antiserum with r alpha z. Affinity purified antiserum strongly recognizes expressed r alpha z, does not recognize r alpha s1, r alpha s1, r alpha o, or r alpha i3, and very weakly interacts with r alpha i1 and r alpha i2. In contrast, the alpha-subunits of purified bovine brain Gi1 and human erythrocyte Gi2 and Gi3 did not react with the alpha z-antiserum. Partially purified mixtures of human erythrocyte G proteins contain a 41-kDa protein that reacts specifically in immunoblots with both crude and affinity purified alpha z-specific antiserum. Quantitative immunoblotting using r alpha z as a standard indicates that there is 60-100 ng of alpha z/micrograms of 40/41-kDa alpha-subunit protein in partially purified human erythrocyte G protein preparations. We conclude that we have identified the alpha z gene product as a 41-kDa trace protein in human erythrocytes.
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Department of Pharmacology, Mount Sinai School of Medicine, New York 10029.
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Iyengar
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D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001923:Brain Chemistry; D002417:Cattle; D003429:Cross Reactions; D004910:Erythrocyte Membrane / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification
D001798:Blood Proteins; C029152:G-substrate; D007106:Immune Sera; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins