Research On S-adenosylmethionine Synthetase And Its Application

S-adenosylmethionine(SAM , AdoMet)is a biologically active moleculardistributed to all body tissues and fluids . It is involved in a number of biochemicalreactions . As methyl group donor , it is important in transmethylation reactionswhich is contributing to the synthesis of such compounds as hormones, nucleicacids, proteins, phospholipids and certain drugs. Moreover, as the precursor ofcertain sulphurated compounds, SAM is concerned with transsulphuration. Bybiotechnological method, SAM synthetase is utilized for the production of SAMusing ATP and L-methionine as substrates, thus SAM synthetase is the key . Matosetc compared the activity of SAM synthetase from different species. The use ofSAM synthetase from E.coli in the synthesis of SAM suffers severe problem inproduct inhibition, thus the SAM synthetase from E.coli is not suitable forlarge-scale synthesis of SAM. As for yeast SAM synthetase, the reaction will takemuch time, and during this time the substrate ATP will decompose , thus we needsupplement ATP in the course of the reaction. The SAM synthetase from rat livermay be a better of choice because of the lowest Km though the specific activity ,thus we choose adult rat liver SAM synthetase for the biotechnological synthesisof SAM.Two forms of SAM synthetase have been identified in the adult rat liver: α? formand β? form . They are isoenzyme. The active forms of α-and β-SAM synthetasesare composed of homotetramers (210 kDa) and homodimers (110 kDa),respectively. Although the α-and β-forms have distinct properties, no differencewas observed in the subunit structure of the α-and β-SAM synthetases. Both α-and β-forms show a single polypeptide band of about 48kDa when analyzed bySDS-PAGE.We accomplished two part of work according to the different resources of SAMsynthetase:(1) Cloning and expression rat liver SAM synthetase in prokaryocytic expressionsystemThis part of wok is to construct SAM synthetase genetic engineering strains for thebiotechnological production of SAM. SAM synthetase cDNA was amplified fromrat liver by RT-PCR technique and inserted into expression vector pQE30. Therecombinant plasmids were transformed into E.coli M15.The subunit molecularweight of SAM synthetase. The synthetase activity was measured by HPLC. Bycontrast with host strains, the enzyme activity of the recombinant strains wasimproved obviously, and this will lay a foundation for the production of SAM bybiotechnological method.(2)Preliminary purification of SAM synthetase from rat liverThe homogenates of rat liver was purified by (NH4)2SO4 fractionation , dialysisand ion-exchange chromatograph on DEAE-Sephacel column. The SAMsynthetase was preliminarily purified and we measured the activity of the enzyme.This will lay a foundation for the further purification and the research of theenzyme .