Patterns of protein synthesis and phosphorylation during anoxia in the land snail Otala lactea

Abstract

Protein labeling patterns with [32P]phosphorous and [35S]methionine plus [35S]cysteine were examined in normoxic and anoxic tissues of Otala lactea. Analysis of protein phosphorylation showed differences between normoxic and anoxic individuals in both the extent of labeling and the pattern of protein phosphorylation. These changes were confined to the hepatopancreas where anoxia caused overall protein phosphorylation to decrease by a factor of 3. Subcellular fractionation of the hepatopancreas showed that this overall decrease in protein phosphorylation was due to a lower level of protein phosphorylation in individual fractions. Thus, the level of protein phosphorylation during anoxia was 45% (plasma membrane fraction), 21% (mitochondrial fraction), 24% (microsomal fraction), and 22% (cytosolic fraction) of that measured in normoxic tissues. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed several changes in protein phosphorylation patterns. For example, the relative phosphorylation of cytosolic proteins of molecular mass 65, 49, 30, 25, 21, and 18 kDa was increased in normoxic individuals. Cytosol from anoxic snails showed a relative increase in the phosphorylation of proteins of molecular mass 68, 52, 44, 37, 18, and 16 kDa. No differences in the protein labeling patterns of the mitochondrial and microsomal fractions were apparent. In contrast, analysis of proteins labeled with [35S]methionine plus [35S]cysteine showed no differences when anoxic and normoxic snail tissues were compared.