N-hydroxysulfosuccinimido active esters and the L-(+)-lactate transport protein in rabbit erythrocytes

Abstract

Esters of N-hydroxysulfosuccinimide strongly inhibit L-(+)-lactate transport in rabbit erythrocytes, probably by acylating amino groups on the transport protein. Lactate transport studies using bis(sulfosuccinimido) suberate (BS3), bis(sulfosuccinimido) adipate (BS2A), bis(sulfosuccinimido) dithiobis(propionate), and a variety of monocarboxylate esters suggest that an exofacial amino group of the lactate transport protein is essential for lactate transport. Also, reductive methylation studies show that even when positive charge is preserved in modified amino groups, the transport is strongly inhibited. At pH less than 6, band 3 mediated inorganic anion transport is enhanced in BS3-treated cells, while at pH greater than 6, it is inhibited. BS3-induced inhibition of L-(+)-lactate transport does not have this pH dependence. BS3 reduces the labeling of a 40-50-kDa membrane polypeptide (band R) by tritiated 4,4'-diisothiocyanato-2,2-dihydrostilbenedisulfonate ([3H]H2DIDS) and by tritiated bis(sulfosuccinimido) adipate ([3H]BS2A). Tritiated sulfosuccinimido acetate (S2[3H]acetate) also labels band R, over a range of concentrations where lactate transport is inhibited in a dose-dependent manner by S2 acetate. BS3 is a known impermeant protein cross-linker. S2 acetate permeates rabbit red cell membranes by an H2DIDS-inhibitable mechanism. BS3 cross-links the proteolytic fragments of rabbit band 3 produced by extracellular chymotrypsin. These labeling experiments support an association between band R and specific monocarboxylate transport.

The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

“…Furthermore, our own data reveal that although the conservative replacement of Arg 306 with lysine allows for expression at the plasma membrane (Figure 6), the protein is catalytically inactive (Supplementary data Table II). This is a similar situation to that found in lactate dehydrogenase where arginine is critical for substrate binding (Hart et al 1987) and like lactate dehydrogenase, MCT1 is inhibited by the arginine-specific agent, phenylglyoxal (Donovan & Jennings 1986, Poole & Halestrap 1993). …”

The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation

“…Furthermore, in the case of rat and rabbit red blood cells, rates of lactate transport were found to be very rapid and to display conventional Michaelis-Menten kinetics indicative of the presence of a single MCT [8,9]. As such, these cells were an ideal starting material for identifying the protein responsible for transport, and several groups used a variety of covalent labelling techniques in their attempts to accomplish this [10][11][12][13]. Unequivocal identification was ultimately achieved in our laboratory using covalent labelling of the protein with 4,4h-di-isothio-cyanatostilbene-2,2h-disulphonate (DIDS) ; labelling was prevented by specific inhibitors of the transporter such as α-cyano-4hydroxycinnamate (CHC).…”

“…A positively charged group that binds the carboxylate anion is a feature that is likely to be present in all MCTs. In red-blood-cell MCT1 arginine probably fulfils this role, since the argininespecific reagent phenyglyoxal inhibits MCT1-mediated lactate transport [2,12,67]. This is also the case in lactate dehydrogenase [68].…”

Identification and partial purification of the erythrocyte l-lactate transporter

“…Jennings & Adams-Lackey (1982) found that incubation of rabbit erythrocytes with [3H]4,4'di-isothiocyanodihydrostilbene-2,2'-disulphonate ([3H]H2DIDS) labelled a membrane protein of 40-50 kDa on SDS/PAGE, in parallel with inhibition of lactate transport. Other reagents which, like H2DIDS, are capable of reacting with exofacial amino groups, afforded protection against labelling of this band by [3H]H2DIDS (Donovan & Jennings, 1985, 1986. However, this evidence for the identity of the transporter is circumstantial, and attempts to label a similar protein in rat erythrocytes, which have a similarly active monocarboxylate transporter to those from rabbit, have met with failure (see ).…”