Probing The Cell Interior With NMR Spectroscopy

Abstract

NMR can give more chemical information than any other non-invasive spectroscopic technique for investigating cell biochemistry (Gadian, 1995; Gillies, 1994). The technique can give information about pathway flux in vivo but rarely gives information about the control exerted by an individual enzyme within a pathway. One way of accessing this information is to titrate the activity of the enzyme of interest with a specific irreversible inhibitor. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of four glycolytic enzymes which together can catalyse the exchange of hydrogen between the C-2 position of lactate and water. We measured this flux in the human erythrocyte using ¹H NMR to measure the rate of ¹H/²H exchange at the C-2 position of L-[2-²H]lactate. By selectively reducing the active concentration of GAPDH in the cell with the irreversible inhibitor iodoacetate and then measuring the dependence of the exchange flux on the active concentration of the enzyme, measured in cell extracts, we were able to determine the specific exchange velocity of the enzyme in the cell (Brindle et al., 1982). This approach is limited, however, by its requirement for membrane permeable and specific irreversible, or quasi-irreversible, inhibitors. The use of molecular genetic techniques to change active enzyme concentration frees us from this restriction. Using these techniques we can, in principle, increase or decrease the concentration of any enzyme or membrane transporter in biological systems ranging from isolated cells to tissues in a whole animal. By measuring the effects of these interventions on an NMR-determined flux we can then measure the kinetic properties of these enzymes or transporters in a completely non-invasive, or at least minimally perturbing, way. For example, in the yeast Saccharomyces cerevisiae the glycolytic enzymes phosphoglycerate kinase (PGK) and GAPDH catalyse the coupled reaction {IE192-1} where GAP is glyceraldehyde 3-phosphate and 1,3-BPG is 1,3-bisphosphoglycerate. This reaction results in exchange of P _i, with the γ-phosphate of ATP. The rate of this exchange, which is of the order of 1-3 mM s⁻¹, can be measured in the intact cell using ³¹P NMR magnetization transfer techniques (Brindle, 1988a). We measured the flux control coefficients of PGK and GAPDH for this exchange by determining the effects of changes in their activity on the exchange flux. The activity of GAPDH was varied by titrating its activity with iodoacetate and the activity of PGK was varied by changing its concentration using molecular genetic techniques. At wild-type levels of the enzymes the flux control coefficient of PGK for the measured flux was about 1. Therefore this flux was a measure of PGK activity in the cell. The kinetic properties of the enzyme in vivo were shown to be similar to those displayed by the isolated enzyme in the test tube. There was no evidence for an alteration in its kinetic properties, as might result, for example, from substrate channelling.