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Protein turnover and stability of the protein pool during metababolic arrest in turtle hepatocytes

University of British Columbia

Hepatocytes isolated from the western painted turtle (Chrysemys picta bellii) are capable of entering a period of metabolic suppression that is characterised by a coordinated and highly regulated reduction in rates of ATP synthesis and ATP demand. To examine the demand side this relationship, the studies described here investigated the partitioning of energy usage in metabolic suppression using protein turnover as an example of a highly regulated and energetically expensive cell process. Absolute rates of protein synthesis fell by 92% during 12h of anoxia at 25°C. Using an empirically determined cost of 5.2 ATPs per peptide bond the relative cost of protein synthesis was determined to be 24.4mol ATP/g/h accounting for 28- 36% of total ATP turnover. In anoxia, this fell to l.6mol ATP/g/h, constituting 25% of total anoxic ATP turnover. The energy dependence of proteolysis was assessed in labile and stable protein pools. During anoxia, labile protein half-lives increased from 24.7 to 34.4h, with stable protein half-lives increasing from 55.6 to 109.6h. Inhibitors of energy metabolism revealed that a large proportion of whole cell proteolytic rates was ATP-independent, with the majority of ATP-dependent proteolysis appearing in the stable protein pool. Consequently, the combined anoxic mean proteolytic suppression for both pools was 36%, but 93% of the ATP-dependent component was suppressed. ATP demand for normoxic ATP-dependent proteolysis was determined at 11. lμmol ATP/g/h, accounting for 21.8% of total ATP- turnover. In anoxia, this was suppressed by 93% to 0.73μmol ATP/g/h accounting for 12% of remaining ATP-turnover. Summation of anaerobic energy demand by proteolysis and protein synthesis accounts for about 40% of remaining ATP-turnover in metabolic suppression. The final series of experiments tested the hypothesis that a heme-protein oxygen sensor is involved in modulating protein expression profiles. Cells incubated in anoxia consistently expressed proteins of 83, 70.4, 42.5, 35.3 and 16. lkDa and suppressed proteins of 63.7, 48.2, 36.9, 29.5 and 17.7kDa. Except for the 70.4 kDa protein, this expression was not found during aerobic incubation with cyanide, used as a mimic of physiological anoxia. Incubation of cells with factors that affect protoporphyrin conformation ( Co⁺²,Ni⁺², CO) produced predictable changes in expression profiles for 42.5, 35.3, 17.7 and 16.1 kDa protein bands and incubation with a heme-synthesis inhibitor abrogated the response. Remaining suppressed proteins in anoxia demonstrated a predictable sensitivity to Co⁺²and Ni⁺²,but no effect with CO, possibly suggesting control by a protoporphyrin with different 0₂ and CO binding kinetics. These results strongly suggest that one or more heme-protein based oxygen sensor mechanisms are present in turtle hepatocytes, which govern both positive and negative modulation of oxygen-sensitive protein profiles in anoxia. Overall, these results demonstrate coordinated reductions in ATP demand by protein turnover, but also demonstrate that protein turnover rates constitute a large proportion of remaining ATP-turnover in anoxic metabolic suppression. An important signal for positive and negative changes in protein expression profiles on the anoxic transition appears to be oxygen itself, raising the suggestion that oxygen-sensing mechanisms may be important in the modulation certain cellular events during metabolic suppression.

Thesis/Dissertation

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2009-06-04

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http://hdl.handle.net/2429/8804

Zoology

University of British Columbia

UBCV

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