Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter

Abstract

In cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue, the transport of D-[3H]adenosine followed Michaelis-Menten saturation kinetics, with Km values of 1.5 +/- 0.3 microM and 1.9 +/- 0.2 microM, respectively. The transport of the isomer, L-[3H]adenosine, showed sigmoidal kinetics in both preparations. In plasma membrane vesicles the S0.5 was 2.5 +/- 0.2 microM with a Hill coefficient of 2.8 and the Vmax value of 0.26 +/- 0.01 pmol s-1 (mg of protein)-1. In cultured chromaffin cells the kinetic parameters for L-[3H]adenosine were S0.5 = 6.2 +/- 0.2 microM and a Vmax 19.7 +/- 0.5 pmol/min per 10(6) cells, with a pronounced positive cooperativity. The Hill coefficient was 4.9. The transport of the L-isomer in cultured cells followed Michaelis-Menten kinetics at the lowest concentrations employed, below 2 microM. On the basis of these results, we propose a kinetic model whereby the adenosine transporter functions mnemonically.

Nucleoside transporter and nucleotide vesicular transporter: Two examples of mnemonic regulation

“…But in the intermediate range of substrate concentrations, a remarkable positive cooperativity with a Hill coefficient of 4.5 ± 0.75 was noted. The mnemonic nucleoside transporter previously described in chromaffin cells also showed high positive cooperativity (Hill coefficient = 4.9) and kinetic parameters similar to those found in endothelial cells for L-adenosine transport [Casillas et al, 1993b].…”

“…More recent studies have shown that besides allosteric modulation by intracellular effectors, such as ATP, the adenosine transporter in chromaffin cells is regulated by extracellular levels of its own substrate, adenosine. This has led to the proposition of a mnemonic model for the es-nucleoside transporter in which kinetic and allosteric cooperation coexist [Casillas et al, 1993b]. The question arises whether allosteric modulation can be a general property of nucleoside transporters.…”

“…Although the nucleoside transporter has been shown to be highly stereoselective, with a strong preference for the D-enantiomer of adenosine [Plagemann et al, 1988], L-adenosine was incorporated efficiently into chromaffin and endothelial cells from adrenal medulla via the NBTI-sensitive equilibrative nucleoside transporter; in contrast to the situation with the D-isoform, however, L-adenosine was not metabolized [Casillas et al, 1993b].…”

Evidence that Adenine Nucleotides Modulate Nucleoside‐Transporter Function

“…The loss of the kinetic cooperativity of the transporter in plasma membrane preparations has been reported [24].…”

“…The uptake of ['Hluridine was measured at 37°C by a stop-filtration technique, a modification of the method used for adenosine [24]. To determine nucleoside transport, a 40-ml portion of vesicle preparation (250 pg protein) was incubated for 1 min at 37 "C and transport was initiated by adding 10 pl calcium-free Locke's solution containing 2 pCi ['Hluridine and non-labeled compound to reach the required concentrations.…”