Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

Abstract

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal :arg stoichiometry of 2 :1 . Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra-and intracellular pH were above 10 . Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium . The rate and specificity of modification depend on the extracellular chloride concentration . At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg . After cleavage of band 3 with extracellular chymotrypsin, [14 C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide . In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments . The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3 H]DIDS to phenylglyoxal-modified membranes was strongly delayed . At DIDS concentrations below 10 j,M, only 50% of the band 3 molecules were labeled with [ 3H]-DIDS during 90 min at 38°C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules .

Nitrate Absorption by Corn Roots

“…As a result, only the essential residue(s) were modified, although there are several other arginyl residues in the protein (2,5,30). Similar results were obtained with human red cells where phenylglyoxal strongly inhibited chloride transport and yet only one arginyl residue was modified per anion transporter (3). Thus, in keeping with the available evidence, the results on nitrate uptake in corn roots suggest the involvement of phenylglyoxal-accessible arginyl residue(s) in nitrate transport through the plasma membrane.…”

“…The apparently covalent nature of the reaction of phenylglyoxal and FITC with amino acid residues should make it possible to label the anion transporting polypeptides in corn roots and identify them by gel electrophoresis. FITC and radiolabeled phenylglyoxal have both been successfully used to specifically label and identify the anion transporter of human red blood cells (3,9). When both intra-and extracellular pH was alkaline, an indiscriminate labeling with [14C]phenylglyoxal was observed (3).…”

“…Phenylglyoxal, a diketone that binds the guanidinium group of arginine (30), strongly inhibited chloride transport in mammalian red blood cells (3). Anion transport in red blood cells was also inhibited by the disulfonic acid stilbene and eosin derivatives, both of which bind the essential lysyl residue of the band 3 protein, which composes the anion transporter (17,18,22,25).…”

“…Likewise, DIDS,3 a disulfonic acid stilbene derivative, inhibited chloride uptake in corn roots and Chara (16,19,20). 14C-Phenylglyoxal, tritiated DIDS, and FITC were successfully used to specifically label and identify the anion transporter in red blood cells (3,9,29). Inhibition of chloride uptake by DIDS in corn roots and Chara was reversible, however, such that radiolabeled DIDS could not be used to label the chloride transporter protein of plant cells (16,19 of Cataldo et al (7).…”

Induction of Nitrate Transport in Maize Roots, and Kinetics of Influx, Measured with Nitrogen-13

“…Like barley, the roots of maize are known to require previous contact with NO3-for full induction of NO3-transport (1 1,16,20), with loss of transport ability during periods of NO3-deprivation in excess of 24 to 48 h (20). Induction of N03-transport has been closely associated with a distinct group of newly synthesized polypeptides in the plasma membrane (4).…”