DE-NOVO PROTEIN-BIOSYNTHESIS RESPONSES TO WATER STRESSES IN WOOD FROGS - FREEZE-THAW AND DEHYDRATION-REHYDRATION

Protein biosynthesis responses occurring during the postthaw period (a fter 12 h freezing at -1.4 degrees C), dehydration (to 27 or 40% of to tal body water lost), or rehydration (after the loss of 40% of body wa ter) were monitored in tissues of spring-collected wood frogs (Rana sy lvatica) after intraperitoneal injection of S-35-labeled methionine cysteine. All six organs tested accumulated radiolabeled amino acids a nd organs of both thawing and rehydrating frogs held at 3-5 degrees C showed a linear increase in amino acid incorporation into the acid-pre cipitable 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 su ppression 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 S-35-lab eled proteins and enhanced labeling of others in extracts from experim ental animals, compared with controls. In particular, new synthesis of proteins with isoelectric points of about 6.0 was prominent and label ed proteins in this IEF peak persisted at 5, 10, or 24 h postinjection , becoming proportionally more important over time. SDS-PAGE analysis of the pI 6.0 peaks from thawed, dehydrated, and rehydrated frogs reve aled the presence of one major low molecular weight protein in each ca se with molecular masses of 15, 13, and 21 kDa, respectively. These da ta indicate that the biochemical adaptations supporting freeze toleran ce and dehydration tolerance in anurans include the stress-induced bio synthesis of a suite of proteins including the novel synthesis of sele cted specific proteins. These proteins may represent stress-related (o r shuck) proteins or may have specific roles in metabolic adaptation i n each state such as in water and ionic balance or cell volume regulat ion. (C) 1997 Academic Press.