Structure and expression of the cDNA for the C isozyme of phosphofructo-1-kinase from rabbit brain.
Rabbit brain contains three phosphofructo-1-kinase (PFK) isozymic subunits designated A, B, and C. The primary structures of the first of these two isozyme types have been determined previously. The isozyme C of rabbit brain was isolated by immunoaffinity chromatography and subjected to proteolytic and chemical digestion. A large number of peptides were sequenced, the total number of amino acids identified being equal to about 80% of the total structure. The sequence of the cDNA derived from brain mRNA for C isozyme was determined from polymerase chain reaction fragments synthesized using oligonucleotides designed on the basis of the peptide sequences. The deduced size of the C isozyme was 86,371 Da, slightly larger than PFKs described previously. The amino acid sequence identity with the rabbit A isozyme was 68.9% and a range of identity to other sequenced mammalian PFKs was 67-69%. Using these data plus previously published data on chemical modification, assignments of the 6 organic ligand binding sites of PFK were inferred. The full-length cDNA was cloned into and expressed in Escherichia coli. Phosphofructokinase C was purified to homogeneity from the bacterial extracts.
Li
Y
Y
Department of Biological Chemistry, University of Health Sciences, Chicago Medical School, Illinois 60064.
Valaitis
A P
AP
Latshaw
S P
SP
Kwiatkowska
D
D
Tripathi
R L
RL
Campbell
M C
MC
Kemp
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010732:Phosphofructokinase-1