Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation

The wood frog, Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state. MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 °C acclimated), 24 h frozen at −2.5 °C, 4 or 8 h thawed at 5 °C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different Km and Vmax values compared with controls; e.g., Km Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas Vmax decreased by 40%. After thawing, Km was restored and Vmax rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased Vmax implicating reversible phosphorylation in MCP regulation during freeze–thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze–thaw.