Interaction of the fluorescent probe, 1‐anilino‐8‐napthalene sulfonate, with the sulfate transport system of ehrlich ascites tumor cells

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Abstract
The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculated the apparent dissociation constant of ANS‐membrane complex (Kd = 6.53 × 10−5 M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight).Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 × 10−3 M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 × 10−6 M) and (3) the transport system has a higher affinity for ANS than for sulfate.These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.