Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate.
The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.
Beaumier
B
B
Laboratoire de Membranologie Moléculaire, Département de Biochimie, Université du Québec à Montréal, Canada.
Béliveau
R
R
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology
D000596:Amino Acids; D002352:Carrier Proteins; D006898:Hydroxylamines; D010710:Phosphates; D019811:Hydroxylamine; D012964:Sodium; D005947:Glucose; D004047:Diethyl Pyrocarbonate