Biomaterial Database

Purification and photoaffinity labeling of sucrose phosphate synthase from spinach leaves. 1990

M E Salvucci, and R R Drake, and B E Haley

U.S. Department of Agriculture, Agricultural Research Service, University of Kentucky, Lexington 40546.

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.

UI	MeSH Term	Description	Entries
D007526	Isoelectric Point	The pH in solutions of proteins and related compounds at which the dipolar ions are at a maximum.	Isoelectric Points,Point, Isoelectric,Points, Isoelectric
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D005964	Glucosyltransferases	Enzymes that catalyze the transfer of glucose from a nucleoside diphosphate glucose to an acceptor molecule which is frequently another carbohydrate. EC 2.4.1.-.	Glucosyltransferase
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D001386	Azides	Organic or inorganic compounds that contain the -N3 group.	Azide
D014532	Uridine Diphosphate Glucose	A key intermediate in carbohydrate metabolism. Serves as a precursor of glycogen, can be metabolized into UDPgalactose and UDPglucuronic acid which can then be incorporated into polysaccharides as galactose and glucuronic acid. Also serves as a precursor of sucrose lipopolysaccharides, and glycosphingolipids.	UDP Glucose,UDPG,Uridine Diphosphoglucose,Diphosphate Glucose, Uridine,Diphosphoglucose, Uridine,Glucose, UDP,Glucose, Uridine Diphosphate
D014539	Uridine Diphosphate Sugars	Nucleotide-sugars such as uridine-diphosphate glucose or UDP-glucose.	UDP Sugars,Diphosphate Sugars, Uridine,Sugars, UDP,Sugars, Uridine Diphosphate
D014675	Vegetables	A food group comprised of EDIBLE PLANTS or their parts.	Vegetable