Primary structure and expression of a human CTP:phosphocholine cytidylyltransferase.
Human CTP:phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library. Initially two degenerate oligonucleotide primers derived from the sequence of the rat liver CT cDNA were used to amplify a centrally located 230 bp fragment. Subsequently overlapping 5' and 3' fragments were amplified, each using one human CT primer and one vector-specific primer. Two cDNAs encoding the entire translated domain were also amplified. The human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary). The region which deviates most from the rat liver CT sequence is near the C-terminus, where 7 changes are clustered within 34 residues (345-359), of the putative phosphorylation domain. The region of the proposed catalytic domain (residues 75-235) is 100% identical with the rat liver sequence. Significant homology was observed between the proposed catalytic domain of CT and the Saccharomyces cerevisiae MUQ1 gene product, and between the proposed amphipathic alpha-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein. There are several shared characteristics of these amphipathic helices. An approx. 42,000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones. The specific activity of the HCT in COS cell homogenates was the same as that of analogously expressed rat liver CT. The activity of HCT was lipid dependent. The soluble form was activated 3 to 4-fold by anionic phospholipids and by oleic acid or diacylglycerol-containing PC vesicles.
Kalmar
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Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Kay
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RJ
LaChance
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Cornell
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D017931:DNA Primers; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase