Cancer cell energy metabolism is characterized by a high glycolytic rate, which is maintained under aerobic conditions. In Ehrlich ascites tumour cells, the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the powerful activator of 6-phosphofructo-1-kinase, is tenfold increased. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), synthesizing and degrading Fru-2,6-P2, was characterized. The molecular mass is 120 kDa. The dependence of PFK-2 activity on the substrate concentrations is hyperbolic (Km for Fru-6-P = 0.09 mM; Km for ATP = 0.7 mM), while the dependence of the FBPase-2 activity on the concentrations of Fru-2,6-P2 is sigmoidal (K0.5 for Fru-2,6-P2 = 4 microM). The PFK-2/FBPase-2 activity ratio is 1. PFK-2 activity is inhibited by citrate (I0.5 = 0.17 mM) and phosphoenolpyruvate (I0.5 = 0.08 mM) but only weakly by glycerol 3-phosphate (I0.5 = 1.57 mM). In contrast to the liver enzyme, the activity of tumour PFK-2/FBPase-2 is not influenced by the action of cAMP-dependent protein kinase. The kinetic properties as well as ion-exchange chromatography pattern differ from their normal counterparts in liver and muscle. The properties are likely to contribute to the maintenance of the high glycolytic rate in these tumour cells.
The flux rates of Pi from blood plasma into the extracellular and intracellular space of mouse liver have been measured in. vivo with the [32P]Pi-tracer technique. The specific radioactivities of plasma Pi and total liver Pi have been measured 0.1-4 min after injection of carrier-free[32P]Pi. The results have been treated with the analogue computer technique. The main conclusions are :1. The flux rate of Pi from blood plasma into the extracellular liver space amounts to 1.14 pmoles and that from the extracellular into the intracellular liver space to 0.39 pmoles per g fresh liver per min.2. The influx of Pi through the liver cell membrane takes place without transient transformation into another phosphate compound rapidly exchangeable with large intracellular phosphate pools.3. The specific radioactivity of intracellular Pi is somewhat higher than that of [y-32P]ATP, thus indicating the absence of intracellular Pi-compartmentation with isotope kinetic relevance.I n previous papers [l--31 the turnover of phosphorylated metabolites within the Embden-Meyerhof pathway and the mononucleotide pool of the normal mouse liver was measured in wivo with the 32P tracer technique. I n these experiments the intracellular liver Pi was omitted since no reliable information was available about the specific radioactivity of metabolically active intracellular Pi. This lack prompted us to elaborate methods for the determination of the extra-and intracellular Pi-turnover in order to obtain more information about its functional behaviour.By tracer flow analysis the following parameters were determined after intravenous injection of carrier-free [32P]Pi: (a) Pi exchange rate between blood plasma and extracellular liver space; (b) Pi exchange rate between extra-and intracellular liver space; (c) Size of the extracellular liver Pi space, which differs considerably from the chloride space; (d) The time course of the specific radioactivity of extra-and intracellular liver Pi.
Methods are described for the rapid determination of free ATP and ATP loosely-and firmlybound to oxygenated hemoglobin using anion exchange resin and ultrafiltration. I n hemolyzates of human erythrocytes under simulated in vivo conditions about 200/, of total ATP is firmly-bound t o hemoglobin with an association constant of about 1 x lo8 (M-1).Depending on the total ATP concentration used, equilibrium between free and firmly-bound ATP is reached within 0.2 and 0.5 sec after ATP addition to "stripped" hemolyzates. I n equilibrium the exchange between these two fractions is very slow, not reaching radioactive equilibrium until several days after labelling the free ATP fraction. On the other hand, exchange between free and loosely-bound ATP is very fast.I n addition t o the known two binding sites for ATP in oxy-and deoxyhemoglobin there exists a third binding site which firmly binds ATP and other anions independent of the oxygenation state of the hemoglobin molecule.I n the last few years an increasing number of papers concerning the binding of anions to hemoglobin [I-51 has appeared. By and large the results reported by different authors are in agreement qualitatively. However, quantitatively the results present certain contradictions. This seems to be due mainly to the methods applied, which do not allow direct differentiation between a t least two classes of binding sites having quite different association constants. Indirect differentiation is possible using various graphic methods available. However, under certain conditions the results obtained in this way are unclear [6]. A second difficulty, which may possibly lead to lowered binding values, could be inefficient "stripping" methods to render hemolyzates phosphate free.I n the course of a tracer kinetic analysis of glycolysis in intact human erythrocytes we were especially interested in definite binding data for ATP t o human hemoglobin. No compatibility with the tracer data could be achieved using the binding data available in the literature. ATP binding was therefore reexamined under simulated in vivo conditions, i.e. 5mM oxygenated hemoglobin, p H 7.2, 37" and intracellular ion concentrations. Furthermore, for the evaluation of tracer data the exchange rates between free and loosely-bound and between free and firmly-bound ATP were required but had not been reported in the literature hitherto. The present paper describes the methods elaborated (a) for rendering hemolyzates completely free of total phosphate, (b) for the rapid and direct determination of ATP in the free form and looselyand firmly-bound to hemoglobin, and (c) for measurements of the exchange rates between free ATP and these two classes of bound ATP. The methods involve ultrafiltration, anion exchange resin treatment and the employment of [y-32P]ATP. Some binding and exchange data for simulated in vivo conditions are reported. EXPERIMENTAL PROCEDUREChemicals ATP, ADP and AMP were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), and purified by paper chromatography according to ...
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