De novo protein biosynthesis responses to water stresses in wood frogs: freeze-thaw and dehydration-rehydration.

ABSTRACT

Protein biosynthesis responses occurring during the postthaw (after 12 h freezing at -1.4 degrees C), dehydration (to 27 or 40% of total body water lost), or rehydration (after the loss of 40% of body water) were monitored in tissues of spring-collected wood frogs (Rana sylvatica) after intraperitoneal injection of 35S-labeled methionine + cysteine. All six organs tested accumulated radiolabeled amino acids and organs of both thawing and rehydrating frogs held at 3-5 degrees C showed a linear increase in amino acid incorporation into the acid-precipitable protein fraction over time. By contrast, dehydrating animals showed little or no increment in protein bound radioactivity over the course of the stress, a result that may be indicative of metabolic suppression in organs when dehydration became severe. Isoelectrofocusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize the proteins synthesized by liver under each experimental state. IEF revealed both new peaks of 35S-labeled proteins and enhanced labeling of others in extracts from experimental animals, compared with controls. In particular, new synthesis of proteins with isoelectric points of about 6.0 was prominent and labeled proteins in this IEF peak persisted at 5, 10, or 24 h postinjection, becoming proportionally more important over time. SDS-PAGE analysis of the p(I) 6.0 peaks from thawed, dehydrated, and rehydrated frogs revealed the presence of one major low molecular weight protein in each case with molecular masses of 15, 13, and 21 kDa, respectively. These data indicate that the biochemical adaptations supporting freeze tolerance and dehydration tolerance in anurans include the stress-induced biosynthesis of a suite of proteins including the novel synthesis of selected specific proteins. These proteins may represent stress-related (or shock) proteins or may have specific roles in metabolic adaptation in each state such as in water and ionic balance or cell volume regulation.