Abstract
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.
Highlights
Interferonsinduceanumberofdifferentproteins ficity) and cellular location of Mx prowhich mediate the antiproliferative, antiviral, and im- tein and the type of virus and its site of replication [5, 6,9]
Munomodulatoryfunctionsofinterferons.Interferon- Murine Mxl protein, which has a distinct nuclear targeting induced M x proteins, which confer resistance to influ- signal (10, 111,can inhibit the replication of influenza, vesicular stomatitis, and measles viruses, containenza virus at a transcriptional level [7,8].Human MxA protein, consensus GTPasesequenceelements.Insectcell-pro- instead, is located in thecytoplasm of the cell, and it can inhibit duced purified murine Mxl andhuman MxA proteins the replication of influenza, vesicular stomatitis, andmeasles were found to hydrolyzeGTP with K,= 65 p~
At least three different inter- homology to each otherand practically no homology to dyferon-induced cellular enzymes, oligo(A) synthetase, p68 pro- namins and VPS1, which suggests that M x proteins and dytein kinase, andMx proteins havebeen shown to be involved in namins most likely have different cellular functions
Summary
Port of newly synthesized proteins, and themyay take part in the regulation of cell differentiation and proliferation. The abbreviationsused are: IFN, interferon;PAGE, polyacrylamide electrophoresis; BSA, bovine serum albumin; GAP, GTPase activating leads to a conformationalchange and the activation of the protein.GTP is hydrolyzed either by theintrinsic GTPase protein; GNRP, guanine nucleotide release protein; GTPyS, guanosine activity of the protein or by the aid of another heterologous. The rate of GTP hydrolor by the aid of a guanine nucleotide release protein (GNRP). This leavesthe protein intoan "empty" state and allows a new GTP molecule to bind into the empty GTP binding site. The GTP binding a n d GTPase activities of different membersof the GTPase superfamily vary considerably (20,21e).,g.
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