ROLE OF MEMBRANE-BOUND 5'-NUCLEOTIDASE IN NUCLEOTIDE UPTAKE BY THE MODERATE HALOPHILE VIBRIO-COSTICOLA

Intact cells of V. costicola hydrolyzed ATP, ADP and AMP. The membrane-bound 5''-nucleotidase (EC 3.1.3.5) was solely responsible for these activities, as shown by experiments with anti-5''-nucleotidase serum and with the ATP analog, adenosine 5''-(.beta..gamma.-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5''-nucleotides and adenosine. The uptake of ATP, ADP and AMP (but not the adenosine uptake) was inhibited by adenosine 5''-(.beta..gamma.-imido)-diphosphate similarly to the inhibition of the 5''-nucleotidase. The uptake of nucleotides had Mg2+ requirements similar to those of the 5''-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [.alpha.-32P]ATP and the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. The adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5''-nucleotidase.