Characterization of human UMP-CMP kinase enzymatic activity and 5' untranslated region.
We have cloned a cDNA for human UMP-CMP kinase from a macrophage cDNA library. Sequence analysis showed that this cDNA is derived from the same gene as a previously reported EST-derived cDNA. Here we show that a conspicuous difference between these two clones, 73 additional 5' nucleotides in the EST clone, including a putative translational start site, is not functionally significant. This work shows that the additional 5'sequence in the EST clone was unnecessary for enzymatic activity and nonfunctional in the initiation of translation. Specifically, we found that protein expressed by both the macrophage-derived cDNA and the extended cDNA had the same relative molecular mass, consistent with use of an ATG internal to the macrophage-derived clone as the functional start site. In addition, this work more precisely defines the catalytic activity of UMP-CMP kinase. Here, we show a 3-fold greater substrate preference for CMP relative to UMP, identify ATP and UTP as the preferred phosphate donors for the reaction, and demonstrate that the reaction is Mg2+-dependent. In addition, investigation of UMP-CMP-kinase expression revealed two mRNA products in immune tissues and cancer cell lines. The smaller RNA product was previously undescribed.
Pearman
A T
AT
Huntsman Cancer Institute, University of Utah, Salt Lake City 84112, USA.
Castro-Faria-Neto
H C
HC
McIntyre
T M
TM
Prescott
S M
SM
Stafforini
D M
DM
D020121:5' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells / Q000166:cytology / Q000201:enzymology; D003001:Cloning, Molecular; D003568:Cytidine Monophosphate / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D015723:Gene Library; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection; D014542:Uridine Monophosphate / Q000378:metabolism
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D014542:Uridine Monophosphate; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D003568:Cytidine Monophosphate