Kinetic assay for measurement of enzyme concentration in situ

摘要: Metabolic pathway analysis requires accurate data regarding enzyme kinetic parameters and metabolite concentrations inside living cells. Intracellular concentration is a critical parameter that notoriously difficult to obtain. We propose method, based on addition, quantify the amount of specific enzymes in metabolic pathways. The known for vast majority were obtained vitro with purified enzymes.More recently, emergence sequenced genomes, use recombinant proteins as sources are now common practice, replacing laborious purification from original organism by production large amounts protein suitable host such Escherichia coli. However, this approach poses major problems analysis. Post-translational modifications not likely occur, protein–protein interactions may be absent. Better strategies clearly needed investigating way more closely resembling vivo state. Recently, noninvasive techniques fluorescence spectroscopy nuclear magnetic resonance (NMR) assuming greater importance but still endure serious limitations [1,2]. Fluorescence restricted few metabolites, namely NAD(P)H, or probes introduced nondestructive In NMR shows great promise hampered its limited time resolution sensitivity. Moreover, intact systems do allow good control individual substrate reaction conditions pH. An alternative study situ [3]. This invasive technique selective cell membrane permeabilization low-molecular weight mole-