Subcellular localization of enzyme activities in chemostat-grown murine myeloma cells

An integrated analysis of enzyme activities, cofactor pools and metabolic fluxes in baculovirus-infected Spodoptera frugiperda Sf9 cells

“…Three ICDH isoforms (a mitochondrial NAD + -dependent and two NADP + -dependent, one cytosolic and another mitochondrial, isoforms) have been described in mammals which differ in cofactor specificity, metabolic function and subcellular distribution. The cytosolic isozyme is involved in the production of NADPH while mitochondrial isozymes are players in the TCA cycle (Caspi et al, 2010;Vriezen and van Dijken, 1998b). In Sf9 cells, we only detected the NADP + -dependent ICDH activity, which in mammalian tissues has an important role in supplying NADPH for cholesterol and fatty acid synthesis and desaturation and for the regeneration of antioxidants, slowing oxidative damage and cellular aging processes (Jennings et al, 1994).…”

Metabolic Flux Analysis of Mammalian Cells

“…Enzyme activity measurements indicate that in mammalian cells probably the aspartate-malate shuttle is the active shuttle system. 38 Another compound that cannot cross the mitochondrial membrane is acetyl-coA. For lipid synthesis, which takes place in the cytosol, acetyl-CoA is transported across the membrane in the form of citrate as described before.…”

“…It is however possible to separate mitochondria experimentally from the cytosol, making direct measurements of, for example, enzyme activity in both compartments possible. 38 …”

“…Vriezen et al 18 measured the activities for different enzymes in SP2/0-Ag14 cells as well as the sub-cellular localization 38 of some enzymes in combination with flux analysis. Their main conclusion was that regulation of metabolism in central metabolism occurs through modulation of enzyme activity and to a much lesser extent through enzyme level.…”

Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells

“…In principle, enzyme activities can be determined either by direct assays in which the enzyme-catalyzed reaction is monitored by measuring product accumulation or substrate depletion over time (Neermann and Wagner, 1996;Vriezen and van Dijken, 1998), or by indirect assays, such as enzymatic cycling systems that increase significantly the sensitivity of enzymatic assays (Gibon et al, 2004;Lowry, 1980). In case changes in product or substrate concentrations cannot be observed directly, it is common practice to use one or more coupling enzymes to generate detectable amounts of product.…”