Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli.
UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.-L. & Chang, H.-Y. (1993) FEBS Lett. 329, 153-158] and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration.
Duggleby
R G
RG
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane, Australia.
Chao
Y C
YC
Huang
J G
JG
Peng
H L
HL
Chang
H Y
HY
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D007763:Lac Operon; D008099:Liver / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009928:Organ Specificity; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase