Purification and Characterization of Sucrose Phosphate Synthase from Sweet Photato Tuberous Roots

Abstract

Sucrose phosphate synthase (SPS) is one of the key enzymes in the sucrose biosynthesis pathway. SPS was purified 40 fold from crude extract of sweet potato tuberous roots by the methods of batch elution from DEAE-Sephacel, PEG precipitation, ω-aminohexyl Sepharose 4B affinity and Mono Q anion exchange chomatographies. The native- and SDS-PAGE analyses revealed SPS to have a native molecular mass of about 540kDa, and it may therefore be homotetramer composed of subunit with a mass of 130-140kDa. The isoelectric point of the purified enzyme as determined by IEF was 5.29. SPS from the sweet potato tuberous root, which differs from the SPS of photosynthetic tissues, was not allosterically regulated by G6P and Pi. The Km for F6P and UDPG was 5.3 and 31.3mM, respectively. The enzyme was activated by Mn (superscript 2+), Mg (superscript 2+), and Ca (superscript 2+), while being inhibited by Hg (superscript 2+). The nucleotides AMP, ADP, ATP, UMP, UDP, UTP, and TDP inhibited the enzyme about 30~50%. The enzyme was sensitive to sulfydryl reagents, but activity could be restored with DTT or β-ME. The enzyme was activated by glucose, glucosamine, maltose, and lactose, but was inhibited by δ-gluconolactone. SPS could also be inhibited by PCMBS and Cibacron blue F3G-A.