Enzymatic characterization of interferon-induced antiviral GTPases murine Mx1 and human MxA proteins.
Interferons induce a number of different proteins which mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. Interferon-induced Mx proteins, which confer resistance to influenza, vesicular stomatitis, and measles viruses, contain consensus GTPase sequence elements. Insect cell-produced purified murine Mx1 and human MxA proteins were found to hydrolyze GTP with Km = 65 microM (Vmax, 7.1 min-1) and 62 microM (Vmax, 3.1 min-1), respectively. The GTPase activity of Mx1 and MxA proteins was strictly dependent on Mg2+ ions. Murine Mx1 protein was inactivated at 10 degrees C lower temperatures than MxA protein. As analyzed, by filter binding assay, Mx1 protein (at 1 microM) showed a relatively high affinity for GDP (Kd = 1.0 x 10(-7) M) and approximately 340-fold lower affinity for guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) (Kd = 3.4 x 10(-5) M). The Kd values for MxA protein were 2.0 x 10(-7) M for GDP and 5.9 x 10(-6) M for GTP gamma S, showing approximately a 30-fold affinity difference. ATP, UTP, or CTP did not inhibit the Mx protein-dependent GTPase activity, suggesting that Mx1 and MxA proteins are highly specific for guanosine nucleotides. In conclusion recombinant nuclear murine Mx1 and cytoplasmic human MxA proteins show clear differences in their enzymatic activities and nucleotide binding characteristics. How these differences influence their cellular functions and antiviral potential is presently not known.
Melén
K
K
Molecular Biology Program, National Public Health Institute, Helsinki, Finland.
Ronni
T
T
Lotta
T
T
Julkunen
I
I
D000818:Animals; D000998:Antiviral Agents / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004790:Enzyme Induction; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009036:Moths; D064547:Myxovirus Resistance Proteins; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000998:Antiviral Agents; C579071:MX1 protein, human; C579070:Mx1 protein, mouse; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D007372:Interferons; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins