Measurement of Binding of Adenine Nucleotides and Phosphate to Cytosolic Proteins in Permeabilized Rat-Liver Cells

Abstract

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight.2. There is no binding of ATP or inorganic phosphate to cytosolic proteins, either in the presence or in the absence of Mg2+.3. Binding of ADP to cytosolic proteins occurs both in the absence and in the presence of Mg2+. The concentration of binding sites was 0.68 and 0.52 pmol/g dry weight of cells (n = 3 -4) in the absence and presence of Mg2 +, respectively.The corresponding Kd values were 320 pM and 235 pM.It has long been recognised that the amounts of metabolites present in a cell are not necessarily a measure of the thermodynamically active species of the metabolites [I -101. Cori [I] introduced the term compartmentation for this phenomenon. Three major types of compartmentation may be envisaged (see [8, 101): firstly, spatial compartmentation may occur due to the existence of permeability barriers; secondly, compartmentation may be caused by binding of metabolites to proteins [2 -81 and thirdly, compartmentation could in principle be caused by diffusion gradients [7, 81.Since in studying metabolic regulation it is of importance to have exact information on the free concentrations of metabolites, methods have been devised to overcome the problems of compartmentation. The best-known is the metabolite indicator method, which has been used effectively to determine the free NADH/NAD+ and NADPH/NADP+ ratios [2-41 (for a review see [I 11). It should be noted that this method gives no information on the extent of binding of metabolites; this information can be obtained by direct measurement. Biicher and Sies [8], for instance, determined the NADH-binding capacity of the cytosolic proteins by Sephadex G-50 column chromatography of an isolated rat-liver cytosol fraction. However, the binding properties of proteins in a dilute solution may differ from those in the intact cell where protein-protein interactions may play a role.We have recently described a procedure for the preparation of rat-liver cells, the plasma membranes of which were rendered permeable to low molecular weight compounds by treatment with filipin whereas cytosolic proteins rcmaincd in situ [12] (see also Lardy et al. [13]). In the present study we have used this preparation to determine the binding of ATP, ADP and phosphate to cytosolic proteins in rat-liver cells. Abbreviation. Mops, morpholinepropanesulphonic acidEnzyme. Lactate dehydrogenase (EC 1.1.1 27) MATERIALS AND METHODS Isolation of Rut-Liver Cells arid Preparation of Filipiii-Treated CellsRat-liver parenchymal cells were isolated from the livers of 24-h-starved male Wistar rats (200-300 g) using a modification [12] of the method of Berry and Friend [14]. Viability was checked by the Trypan blue exclusion method; at least 90 "/, of the cells excluded the dye.Filipin-treated cells were prepared exactly as described in [12]. They were finally suspended in a standard ...

Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

“…Provided that the number of binding sites is not changed, such a decrease implies an increase of the dissociation constant by a factor of about 2. The difference between our results and those of Gankema et al (1983), however, amounts to a factor of at least 20 and therefore cannot be explained by an effect of poly-(ethylene glycol). Whether the low-affinity binding site found in permeabilized liver cells is an additional cytosolic binding site lost in the present study during affinity chromatography on Ap5A-agarose or whether it represents a membrane-associated binding site cannot be decided on the basis of the available data.…”

“…One of the main differences between the present study and the work of Gankema et al (1983) is the use of poly(ethylene glycol) to induce high local protein concentrations, instead of permeabilized liver cells. In our system, binding of ADP is 36 % lower in the absence of poly(ethylene glycol) than in its presence.…”

“…Using permeabilized rat liver cells, Gankema et al (1983) observed only one binding site for ADP, with a dissociation constant of 320/tM in the absence and 235 /aM in the presence of Mg2". One of the main differences between the present study and the work of Gankema et al (1983) is the use of poly(ethylene glycol) to induce high local protein concentrations, instead of permeabilized liver cells.…”

“…The mitochondrial enzyme may in part be released during tissue homogenization, thereby increasing adenylate kinase activity in isolated liver cytosol. Inhibition of adenylate kinase by EDTA (Gankema et al, 1983) was not feasible, since, as shown below, EDTA itself influenced the binding of ADP. Furthermore, Ap5A, a potent inhibitor of muscle adenylate kinase, could not be used because it is a relatively poor inhibitor of liver adenylate kinase (Feldhaus et al, 1975) and because, as a structural analogue of the adenine nucleotides, it could interfere with the determination ofADP binding.…”

“…In permeabilized rat liver cells, binding of adenine nucleotides has been studied by Gankema et al (1983). About 30 % of the cytosolic ADP was found to be bound at physiological concentrations of ADP, whereas ATP was not bound at all.…”