Effect of arginine modification on K(+)-dependent leucine uptake in brush-border membrane vesicles from the midgut of Philosamia cynthia larvae.
The effect of phenylglyoxylation on the midgut K(+)-dependent leucine transport was studied using lepidopteran brush-border membrane vesicles. The inhibition of leucine uptake by phenylglyoxal (PGO) showed a biphasic inactivation pattern. The second-order rate constant for the slow and fast phases were 0.0020 mM-1 min-1 and 0.0091 mM-1 min-1, respectively. However, substitution of borate buffer for Hepes-Tris buffer produced a mono-exponential inactivation pattern, suggesting modification of a single arginine group. The effect of PGO was dose-dependent and the concentration causing half-maximal inhibition of leucine uptake was 5.1 +/- 0.3 mM. Leucine transport was significantly inhibited also in the absence of a potassium electrochemical gradient (i.e., [K+]in = [K+]out = 100 mM), suggesting that inhibition was not related to a decrease in the driving force. Moreover, intravesicular volume remained unchanged after preincubation with PGO. Kinetic analysis of the interaction of PGO with the leucine cotransporter revealed that (i) inhibition was related to a decrease in the Vmax value and (ii) neither leucine nor K+ were able to prevent the inhibition. Our results suggest an important role for arginine residues in the molecular mechanism of K+/leucine cotransport in lepidopteran larvae midgut.
Parenti
P
P
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Hanozet
G M
GM
Villa
M
M
Giordana
B
B
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D004064:Digestive System / Q000378:metabolism*; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D007915:Lepidoptera; D007930:Leucine / Q000378:metabolism*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D010658:Phenylglyoxal / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*
D001120:Arginine; D007930:Leucine; D010658:Phenylglyoxal; D011188:Potassium