Isolation of a precursor and a nascent chain form of glucose-6-phosphate dehydrogenase from rat uterus and regulation of precursor processing by estradiol

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SDS-polyacrylamide gel electrophoresis of anti-glucose-6-phosphate dehydrogenase immunoprecipitates from radiolabeled uterine tissue extracts previously revealed three proteins: A, B and C, which were tentatively identified as a 60–64 kDa precursor form, a 57 kDa predominant form, and a 40–42 kDa nascent peptide form of the enzyme, respectively. A peptide-mapping technique was used to examine structural homologies among A, B and C. Following the labeling of uterine proteins with [ 35S]methionine, labeled proteins A, B and C were isolated by immunoprecipitation and electrophoresis. Each protein was individually co-digested with authentic, [ 3H]methionine-labeled glucose-6-phosphate dehydrogenase using papain, the resulting peptides were resolved by isoelectric focusing and the peptides from the two sources on each gel were compared using double-label counting methods. Proteins, A, B and C had at least eight peptides in common, both proteins A and c had two additional peptides in common that were not present in proteins B, and B protein had two peptides that were either absent or present in reduced amounts in digests of proteins A and C. The extensive structural homology and immunoreactivity of these proteins indicated that proteins A, B and C are well related to glucose-6-phosphate dehydrogenase. The presence of two extra peptides in proteins A and C suggested that these peptides may be derived from a common NH 2-terminal leader sequence which was present in both the precursor and nascent peptide chains. The presence of two peptides that were present in protein B and absent from proteins A and C is easiest to explain if they are derived from the two ends of the molecule, with the corresponding peptides in proteins A and C containing additional peptide sequence that are ‘normally’ removed by endogenous proteolytic processing enzymes. Based on the relative time-course of synthesis of the three glucose-6-phosphate dehydrogenase-related proteins in control and estrogen-treated uteri, it appears that estradiol promotes an increase in the relative rate of transfer of label from protein A into B by stimulating the rate of processing of the precursor to the predominant form of the enzyme and enhances the rate of translational conversion of protein C into higher molecular weight forms.