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 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.
Al-Robaiy
S
S
Institute of Biochemistry, University of Leipzig, School of Medicine, Germany.
Eschrich
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006597:Fructose-Bisphosphatase