PLANT PHYSIOLOGY , Vol 102, Issue 2 529-536, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Identification of the Uridine-Binding Domain of Sucrose-Phosphate Synthase (Expression of a Region of the Protein that Photoaffinity Labels with 5-Azidouridine Diphosphate-Glucose)
M. E. Salvucci and R. R. Klein
United States Department of Agriculture, Agricultural Research Service, and Agronomy Department, University of Kentucky, Lexington, Kentucky 40546-0076
The uridine diphosphate-glucose (UDP-Glc) binding domain of
sucrose-phosphate synthase (SPS) was identified by overexpressing part of
the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide
primers corresponding to two tryptic peptides common to both the
full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled
with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a
polymerase chain reaction to isolate a partial cDNA clone. Comparison of
the deduced amino acid sequence of spinach SPS with the sequences of potato
sucrose synthase showed that the partial cDNA included one region that was
highly conserved between the proteins. Expression of the partial cDNA clone
of SPS in Escherichia coli produced a 26-kD fusion protein that
photoaffinity labeled with 5-N3UDP-Glc. Photo-affinity labeling of the
26-kD fusion protein was specific, indicating that this portion of the SPS
protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide
from the photolabeled protein provided tentative identification of amino
acid residues that make up the uridine-binding domain of SPS.
