Tyrosine transport in the brush border membrane of larval Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

Young Seok Hong, Purdue University

Abstract

Tyrosine transport across the brush border membrane of Leptinotarsa decemlineata was characterized using brush border membrane vesicles (BBMV). L-tyrosine transport into BBMV was stimulated by both K$\sp+$ and Na$\sp+$ while it was not enhanced by Li$\sp+$, Rb$\sp+$, Cs$\sp+$, Ca$\sp{2+}$ nor Mg$\sp{2+}$. Compared to ion-independent L-tyrosine transport, KSCN and NaSCN increased both the J$\rm\sb{max}$ and K$\rm\sb{t}$ for L-tyrosine uptake. Ion-independent L-tyrosine uptake was strongly inhibited by leucine, phenylalanine, proline, and serine and moderately inhibited by lysine, threonine, tryptophan, and valine. Of 19 amino acids tested only cysteine inhibited the uptake in the presence of NaSCN. The tyrosine transporter was not affected by D-tyrosine. Among potassium salts of anions tested, Cl$\sp-$ and SCN$\sp-$, which more readily cross lipid membranes, increased tyrosine uptake. Less permeant PO$\sb4\sp{2-}$ and gluconate did not affect tyrosine uptake, showing that tyrosine transport in L. decemlineata is rheogenic. cis K$\sp+$ higher or equal to trans concentration was needed for a maximal tyrosine uptake rate in L. decemlineata. cis KSCN stimulated initial tyrosine uptake rate in a concentration dependent-manner, reaching a maximum at 20 mM KSCN. At pH 6.4 and 7.4, tyrosine uptake rates were significantly higher compared to that seen in an alkaline (pH 9.4) uptake medium. There was, however, no difference in tyrosine uptake rates between pH 6.4 and 7.4 conditions. To identify effective inhibitors of the tyrosine transporter, experiments with Bacillus thuringiensis (Bt) $\delta$-endotoxins, amino acid analogs, and plant lectins were conducted. Lectins did not inhibit initial tyrosine uptake, indicating that the active site(s) on the tyrosine transporter is unlikely to have functionally important glycosyl groups tested. Neither MeAIB (methylamino-isobutyric acid) nor BCH (2-aminobicyclo- (2,2,1) -heptane-2-carboxylic acid), inhibitors of amino acid transport systems A and L, respectively, inhibited tyrosine uptake. Although only Bt var. tenebrionis (Btt) toxin is active against L. decemlineata in vivo, both Btt and CryIA(c) toxins decreased the equilibrium level of tyrosine uptake in a concentration-dependent manner. Bt toxins may exert their effects by disrupting the integrity of membrane vesicles, resulting in reduction of total internal volume of BBMV. However, neither Bt toxin affected initial tyrosine uptake rates.

Degree

Ph.D.

Advisors

Neal, Purdue University.

Subject Area

Entomology|Molecular biology

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