FIGURE 3.

Effect of hypoxia on aconitase and RNA binding activity of IRP1^WT and IRP1^S138E expressed in HEK 293 cells. A, calculated distribution of aconitase and RNA-binding forms for WT (n = 3) and S138E (n = 4) in cells grown under normoxic (N) or hypoxic (Hy) conditions. The activities and protein levels are for the tet-induced IRP1; endogenous (–tet) aconitase activities or protein levels have been subtracted. RNA binding was determined using antibody supershift EMSA with the anti-Myc antibody. The mass of the c-acon pool was determined using the established specific activity of purified c-acon, and the mass of the RNA binding pool was determined by using the specific radioactivity of the [³²P]RNA used for EMSA and assumed a 1:1 IRP1/IRE ratio under saturating conditions (see “Experimental Procedures”). Panel i, aconitase activity attributable to IRP1^WT or IRP1^S138E was determined. For normoxic S138E lysate acon activity, the resulting value was below zero, denoted by #. Panel ii, effect of hypoxia on RNA binding activity of IRP1^WT and IRP1^S138E was also determined. Although the mean RNA binding appeared to decrease for IRP1^WT in hypoxia, the difference was not statistically significant. The mean RNA binding activity of IRP1^S138E was not reduced by hypoxia. Panel iii, total active forms of IRP1/c-acon was determined by summing the aconitase and RNA binding activity for the wild type and S138E phosphomutant under each condition using the data in Panels i and ii. Panel iv, IRP1 protein was quantified in lysates by anti-IRP1 immunoblot using purified recombinant IRP1 for the standard curve. An asterisk indicates hypoxia value significantly different from corresponding normoxia control (Student's t test, p ≤ 0.05). B, pie chart representation of the data in A. The percentages of forms were calculated by dividing the active form (pmol/mg lysate protein) by the total IRP1 protein (pmol/mg lysate protein) from the quantitative immunoblot. When activity was below the endogenous background, which occurred only for acon activity of S138E lysates in normoxia, a value of zero percent was assigned. C, freshly prepared lysates from HEK cells were anaerobically treated with DEANO or an equivalent volume of carrier buffer (control). After a 15-min incubation each reaction was quenched in assay mix (0.1 m Tris, pH 8.0, 20 mm dl-isocitrate), and aconitase activity was measured. Endogenous activity (–tet) was subtracted from total activity (+tet) to determine the activity caused by c-acon encoded by the transgene. Starting aconitase activity for c-acon^WT was 40.3 ± 12.4 millinunits/mg protein, whereas for c-acon^S138E it was 8.7 ± 1.8 milliunits/mg protein (means ± S.E., n = 3). An asterisk denotes c-acon^S138E activity significantly lower than corresponding c-acon^WT activity (Student's t test, two-tailed, unpaired, n = 3, p < 0.05). WT, wild type.