Human uridine monophosphate synthase: baculovirus expression, immunoaffinity column purification and characterization of the acetylated amino terminus.
Human uridine monophosphate (UMP) synthase, a bifunctional protein containing orotate phsophoribosyltransferase (OPRTase, EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (ODCase, EC 4.1.1.23) activities, has been overproduced by construction and use of a recombinant baculovirus containing the cDNA for this protein. Expression of the virus in cabbage looper larvae produces a crude larval homogenate having UMP synthase enriched about 180-fold over human placental homogenates and allows larger quantities of this human protein as well as analog proteins to be prepared for structure/function studies. A vastly improved purification procedure using a monoclonal immunoaffinity column was developed. Human UMP synthase purified from larval extracts yielded a product which comigrates in SDS gel electrophoresis with UMP synthase purified from human placenta; pure proteins prepared from these two tissue sources have the same specific activities. We found that OPRTase requires Pi ions in the assay buffers for optimal OPRTase activity; BSA in the assay vessel increases to a lesser degree both OPRTase and ODCase activities. These changes in the assay are essential to observe a parallel enrichment of the two enzyme activities. The baculovirus system was used to express human UMP synthase because it usually yields a product with posttranslational modifications that reflect those of the organism that provided the cDNA. We report data to show that human UMP synthase derived from either human placenta or larval extracts both have a sequence in which the N-terminal methionine has been removed and the formerly penultimate alanine has been acetylated.
Han
B D
BD
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 27599-7260, USA.
Livingstone
L R
LR
Pasek
D A
DA
Yablonski
M J
MJ
Jones
M E
ME
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017924:Nucleopolyhedroviruses; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera
D009097:Multienzyme Complexes; D010710:Phosphates; D011994:Recombinant Proteins; C027157:uridine 5'-monophosphate synthase; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase