TOPOGRAPHIC ANALYSIS OF METABOLIC PATHWAYS IN SINGLE LIVING CELLS BY MULTISITE MICRO-FLUOROMETRY

Multichannel (multisite) microfluorometry in conjunction with microinjection of metabolic intermediates (e.g. glycolytic phosphate esters) was used for in situ topographic analysis of metabolic transients (e.g. NAD(P)NAD(P)H) in correlation to structure and compartmentalization of single living EL2 and L cells. In cells submitted to repeated microinjections with different doses of substrate (glucose-6-P or -1-P, 6-phosphogluconate, allosteric factors) rate laws were derived by a power or exponential approximation. On this basis intracellular metabolic rates were evaluated topographically and the multisite-multicomponent control of enzyme pathways in integrated biochemical systems was assessed. From different simulation trials it appeared that the transients observed are best simulated by a difference of exponentials, accounting for NAD(P) reducing and reoxidizing pathways. The determination of intracellular metabolic rate laws, their multisite-multicomponent control and the extent to which topographic discrimination of compartmentalization is possible provide the basis for application to specific problems in cell physiology, specialized cell function or the understanding of multicellular steady states. © 1979.