Purification and characterization of sucrose phosphate synthase from sweetpotato tuberous roots

Sucrose phosphate synthase (SPS) is one of the key enzymes in the sucrose b iosynthesis pathway. SPS was purified 40 fold from crude extract of sweet p otato tuberous roots by the methods of batch elution from DEAE-Sephacel, PE G precipitation, omega -aminohexyl Sepharose 4B affinity and Mono Q union e xchange chomatographies. The native- and SDS-PAGE analyses revealed SPS to have a native molecular mass of about 540 kDa, and it may therefore be homo tetramer composed of subunit with a mass of 130-140 kDa. The isoelectric po int of the purified enzyme as determined by IEF was 5.29. SPS fi-om the swe et potato tuberous root, which differs from the SPS of photosynthetic tissu es, was not allosterically regulated by G6P and Pi. The Km for F6P and UDPG was 5.3 and 31.3 mM, respectively. The enzyme was activated by Mn2+, Mg2+, and Ca2+, while being inhibited by Hg2+ The nucleotides AMP, ADP, ATP, UMP , UDP, UTP, and TDP inhibited the enzyme about 30 similar to 50%. The enzym e was sensitive to sulfydryl reagents, but activity could be restored with DTT or beta -ME. The enzyme was activated by glucose, glucosamine, maltose, and lactose, but was inhibited by delta -gluconolactone. SPS could also be inhibited by PCMBS and Cibacron blue F3G-A.