Identification of membrane-bound phosphoglucomutase and glucose-6 phosphatase by 32P-labeling of rat liver microsomal membrane proteins with 32P-glucose-6 phosphate.
Three recent reports have suggested that rat liver microsomal glucose-6 phosphatase (Glc6Pase) should be a 62-64 kDa polypeptide. In this work, we examine the possibility that the 62-64 kDa could represent a functional dimeric form of the 36.5 kDa glucose-6 phosphate (Glc6P)-phosphohydrolase, previously identified [Countaway et al. (1988) J. Biol. Chem. 263, 2672-2678]. From 32P-labeling experiments with 32P-Glc6P and analysis of 32P-labeled protein by SDS-PAGE and autoradiography, we show that three different rat liver microsomal polypeptides, the apparent molecular masses of which are 62, 54, and 37 kDa, may be specifically labeled with 32P-Glc6P. We demonstrate that the 62 kDa polypeptide is a microsome-bound form of cytosolic phosphoglucomutase, by combining labeling competition experiments and enzymatic assay. It should likely not account for a putative dimeric form of Glc6P-phosphohydrolase. The 37 kDa polypeptide fulfills the criteria of Glc6P-phosphohydrolase. We have not obtained any definitive evidence for its assembly as a dimer under functioning conditions. The 32P-Glc6P-labeling characteristics of the 54 kDa polypeptide are those expected for a protein displaying affinity in the millimolar range of concentration and a high binding capacity for Glc6P. They are consistent with those of a 54 kDa microsomal polypeptide, previously suggested to be involved in Glc6Pase activity.
Mithieux
G
G
INSERM U. 197, Faculté de Médecine Alexis Carrel, Lyon, France.
Ajzannay
A
A
Minassian
C
C
D000818:Animals; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000032:analysis* / Q000378:metabolism; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010733:Phosphoglucomutase / Q000032:analysis* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D013379:Substrate Specificity
D002793:Cholic Acids; D004220:Disulfides; D005958:Glucosephosphates; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D019298:Glucose-6-Phosphate; D005952:Glucose-6-Phosphatase; D010733:Phosphoglucomutase; D019826:Cholic Acid