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Samiya Al-Robaiy, Klaus Eschrich

Rat Muscle Fructose-1,6-Bisphosphatase: Cloning of the cDNA, Expression of the Recombinant Enzyme, and Expression Analysis in Different Tissues

The 1282 bp cDNA of an isoenzyme of fructose-1,6- bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose- 1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 ?g of total RNA of rat muscle contains 1.7 × 10⁶ molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 × 10⁴ copies of this message were found per ?g total RNA of heart and kidney, respectively.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 380, 09/1999
Pages: 1079 - 1085

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