A heme-protein-based oxygen-sensing mechanism controls the expression and suppression of multiple proteins in anoxia-tolerant turtle hepatocytes.

The O2 sensitivity of protein expression was assessed in hepatocytes from the western painted turtle. Anoxic cells consistently expressed proteins of 83.0, 70.4, 42.5, 35.3, and 16.1 kDa and suppressed proteins of 63.7, 48.2, 36.9, 29.5, and 17.7 kDa. Except for the 70.4-kDa protein, this pattern was absent during aerobic incubation with 2 mM NaCN, suggesting a specific requirement for O2. Aerobic incubation with Co2+ or Ni2+ increased expression of the 42.5-, 35.3-, and 16.1-kDa protein bands which was diminished with the heme synthesis inhibitor 4,6-dioxoheptanoic acid. Proteins suppressed in anoxia were also suppressed during aerobic incubation with Co2+ or Ni2+ but this was not relieved by 4,6-dioxoheptanoic acid. The anoxia- and Co2+/Ni2+-induced expression of the 42.5-, 35.3-, and 16.1-kDa protein bands was antagonized by 10% CO; however, with the exception of the 17.7-kDa protein, this was not found for any of the O2- or Co2+/Ni2+-suppressed proteins. Anoxia-induced proteins were compared with proteins expressed during heat shock. Heat shock proteins appeared at 90.2, 74.8, 63.4, 25, and 15.5 kDa and were of distinct molecular masses compared with the anoxia-induced proteins. These results suggest that O2-sensing mechanisms are active in the control of protein expression and suppression during anoxia and that, in the case of the 42.5-, 35.3-, 17.7-, and 16.1-kDa proteins, a conformational change in a ferro-heme protein is involved in transducing the O2 signal.

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco1

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.