Purification and photoaffinity labeling of sucrose phosphate synthase from spinach leaves.
Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethylene glycol, ion-exchange and hydrophobic interaction chromatography, and rate zonal centrifugation. The enzyme was purified more than 600-fold to a specific activity of 57 mumol/min/mg protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a 120-kDa polypeptide was enriched through purification and was the major polypeptide in the final SPS preparation. The 120-kDa polypeptide was photoaffinity labeled with the substrate analog, 5-azidouridine [beta-32P]5'-diphosphate-glucose ([beta-32P]5-N3UDP-Glc). Covalent incorporation of 5-N3UDP-Glc into the 120-kDa polypeptide exhibited an apparent Kd of 74 microM, similar to the apparent Ki for inhibition of SPS activity by unphotolyzed 5-N3UDP-Glc. Competition experiments showed that photolabeling of the 120-kDa polypeptide by 5-N3UDP-Glc was reduced in the presence of UDP-Glc, exhibiting an apparent Ki value that was similar to the apparent Km (UDP-Glc) of 2.9 mM for the purified enzyme. The relative molecular mass of the SPS holoenzyme was 253,000, and the isoelectric point of the 120-kDa subunit was 5.2. The data confirmed the identity of the 120-kDa polypeptide as the SPS subunit, established the structure of the active enzyme as a dimer, and demonstrated active-site labeling of SPS by a photoaffinity analog of the substrate.
Salvucci
M E
ME
U.S. Department of Agriculture, Agricultural Research Service, University of Kentucky, Lexington 40546.
Drake
R R
RR
Haley
B E
BE
D000345:Affinity Labels; D001386:Azides / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000302:isolation & purification*; D007526:Isoelectric Point; D008970:Molecular Weight; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000494:pharmacology*; D014539:Uridine Diphosphate Sugars / Q000494:pharmacology*; D014675:Vegetables / Q000201:enzymology*
D000345:Affinity Labels; D001386:Azides; D014539:Uridine Diphosphate Sugars; C060227:5-azidouridine 5'-diphosphoglucose; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D014532:Uridine Diphosphate Glucose