Structure, distribution, and functional expression of the phosphofructokinase C isozyme.
To elucidate the structure, tissue-specific expression, and allosteric properties of phosphofructokinase-C (PFK-C), we cloned the cDNA for PFK-C from a rat hypothalamic cDNA library. The cDNA is 2643 base pairs long and encodes a protein of 765 amino acids. The deduced amino acid sequence is highly homologous to PFK-M (muscle) and PFK-L (liver), 69 and 65% amino acid identity, respectively, especially at substrate binding and catalytic sites, while the allosteric binding sites are less conserved. Tissue-specific expression of PFK-C was investigated by Northern blot analysis. PFK-C mRNA was detected in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other tissues. In situ hybridization showed that PFK-C is expressed at a higher level in higher brain regions such as the cortex, compared with the midbrain and basal ganglia, while PFK-L is expressed at approximately equal levels throughout the brain. Expression plasmids containing PFK-C and PFK-L coding sequences were constructed and expressed by transient transfection into CMT cells. Expression of transfected PFKs was demonstrated by PFK enzymatic activity and by Western blotting with anti-rat brain and liver PFK antisera. Allosteric regulatory properties of PFK-C and PFK-L expressed in CMT cells were compared. Fructose 2,6-bisphosphate, a potent activator of PFK, decreased the Km of PFK-C for fructose 6-phosphate from 200 to 60 microM while decreasing that of PFK-L from 300 to 55 microM. The properties of PFK-C and PFK-L expressed in CMT cells clearly demonstrate the allosteric differences between the different PFK isozymes.
Gekakis
N
N
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
Johnson
R C
RC
Jerkins
A
A
Mains
R E
RE
Sul
H S
HS
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D007031:Hypothalamus / Q000201:enzymology*; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009928:Organ Specificity; D010732:Phosphofructokinase-1 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D010732:Phosphofructokinase-1