Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene

Abstract

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

Biphasic effect of extra-reticular Mg2+ on hepatic G6P transport and hydrolysis

“…5. As the figure illustrates, in the presence of alamethicin (3μg/ml) or histone 2A (250μg/ml or isodose vs. the protein content in the incubation system, [9,19]) G6P hydrolysis in the presence of 0.25mM extra-reticular Mg 2+ was significantly lower than that observed in cells permeabilized only with digitonin. Following the addition of alamethicin or histone 2A, a similar amount of Mg 2+ was lost from the ER ( Table 2).…”

“…Histone 2A [19] and alamethicin [9,19] have been used in alternative to taurocholate or deoxycholate to enhance permeability of ER membrane to G6P bypassing the transport mechanism. The effect of either of these agents on G6P hydrolysis is reported in Fig.…”

“…In selected experiments, histone 2A (250μg/ml or isodose with the amount of protein present in the system, [19]) or alamethicin (3μg/ml, [19]) were added directly to the incubation mixture containing digitonin-permeabilized hepatocytes to enhance ER membrane permeability to G6P [9,19]. In a separate set of experiments, the ionophore A23187 was added at the final concentration of 2μg/ml to mobilize Mg 2+ from ER [20] without altering ER membrane permeability to G6P.…”

“…Glucose-6-phosphatase (G6Pase, EC 3.1.3.9) is an enzymatic complex operating within the endoplasmic reticulum (ER) of liver [7], kidney [8] and pancreatic beta-cells [9]. Because it catalyzes the hydrolysis of glucose 6-phosphate (G6P) to glucose and phosphate (Pi), this enzyme has a key role in regulating glycolysis and glucose homeostasis [10].…”

Proinsulin C-peptide potentiates the inhibitory action of insulin on glucose synthesis in primary cultured rabbit kidney-cortex tubules: Metabolic studies

“…Nevertheless, it is still possible that a decreased flux through G6Pase may contribute to the insulin-and C-peptideevoked inhibition of glucose synthesis due to reduced import of G6P by the endoplasmic reticulum (ER) membrane G6P-transporter (G6PT, also known as SLC37A4) (Pan et al 1999). This protein has been immunodetected in kidney microsomes by Senesi et al (2005). A diminished G6P transport into the ER lumen may limit activity of the renal glucose-6-phosphatase-a (G6Pase-a), which exhibits its active site within the ER lumen (Chou et al 2010) and shows to maintain interprandial glucose homeostasis (Chou et al 2013).…”