Inhibition of L-lactate transport and band 3-mediated anion transport in erythrocytes by the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl-4,4'-diaminostilbene-2,2'-disulpho nat e (TBenzDS).
(1) The synthesis of the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl- 4,4'-diaminostilbene-2,2'-disulphonate (TBenzDS) is described, and its interaction with the lactate transporter and band 3 protein of erythrocytes investigated. At 10% haematocrit the IC50 (concn. required for 50% inhibition) for inhibition of transport of 0.5 mM L-lactate into rat erythrocytes at 7 degrees C was approx. 1.6 microM, as low as any other inhibitor of the transporter. In human erythrocytes at 10% haematocrit the IC50 value was increased from approx. 3 microM to 9 microM upon raising the temperature from 7 degrees C to 25 degrees C. (2) TBenzDS inhibited transport of L-lactate into rat erythrocytes in a manner that was competitive with the substrate, as is the case for some other stilbene disulphonate derivatives (Poole, R.C. and Halestrap, A.P. (1991) Biochem. J. 275, 307-312). (3) Increasing the haematocrit from 5 to 20% caused a 3-fold increase in the IC50 value for inhibition of L-lactate transport in rat erythrocytes. (4) TBenzDS was found to bind to erythrocyte membranes, with a partition coefficient (Pm) of 6000-7000 under all conditions tested. (5) TBenzDS also inhibited band 3-mediated sulphate transport in rat erythrocytes; 50% inhibition required approx. 2.5 microM TBenzDS for cells at 10% haematocrit. (6) TBenzDS is fluorescent, and an enhancement of this fluorescence occurs upon addition of BSA or erythrocyte membranes. The fluorescence enhancement caused by erythrocyte membranes is due to binding of the inhibitor to the band 3 protein at the same site as the stilbenedisulphonate 4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS).
Poole
R C
RC
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Cranmer
S L
SL
Holdup
D W
DW
Halestrap
A P
AP
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D001593:Benzyl Compounds / Q000737:chemistry / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D006400:Hematocrit; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D015394:Molecular Structure; D051381:Rats; D013267:Stilbenes / Q000737:chemistry / Q000494:pharmacology*
D001457:Anion Exchange Protein 1, Erythrocyte; D001593:Benzyl Compounds; D007773:Lactates; D013267:Stilbenes; C072037:N,N,N',N'-tetrabenzyl-4,4'-diaminostilbene-2,2'-disulfonate; D019344:Lactic Acid