title: Demonstration and characterization of a transport system capable of lysine and leucine absorption that is encoded for in porcine jejunal epithelium by expression of mRNA in Xenopus laevis oocytes.
abstract: Defolliculated Xenopus laevis oocytes were injected with size-fractionated poly(A)+ RNA (RNA) isolated from the jejunal epithelium of growing pigs (average BW 33.8 kg) to identify proteins capable of Na+ -independent amino acid transport. The ability of oocytes to absorb L-lysine (lysine) or L-leucine (leucine) from Na+- free media was quantified in oocytes after injection of RNA fractions or water. Specific RNA fractions were identified that induced saturable uptake of lysine (Kt = 52 microM) and leucine (Kt = 97 microM), whereas endogenous oocyte uptake was not saturable. Induced uptake of .05 mM lysine by oocytes was inhibited (P < .05) 68.1% by 5 mM leucine and 38.9% by .2 mM L-cystine (cystine). Induced uptake of .05 mM leucine was inhibited (P < .05) 83.1% by 5 mM lysine and 23.2% by .2 mM cystine. Although not significant (P > .05), 5 mM L-glutamate (glutamate) quantitatively stimulated the induced uptake of .05 mM lysine by 18.8% and the induced uptake of .05 mM leucine by 60%. To identify mRNA species responsible for this bo,+ transporter-like activity, oocytes were co-injected with the RNA fractions and degenerate DNA oligomers complementary (antisense) to the cloned human kidney bo,+ amino acid transporter, or (as a negative control) with a DNA oligomer complementary to the rabbit intestinal Na+/glucose cotransporter, or with water. Only those oocytes injected with two specific RNA fractions and the antisense DNA oligomer complementary to the bo,+ transporter displayed reduced (P < .05) uptake of lysine (45.7, 55.4%) and leucine (44.1, 65.9%). These results indicate that messenger RNA encoding for a protein capable of stimulating the competitive absorption of lysine and leucine is expressed by the jejunal epithelia of growing pigs.
authors.affiliation: Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Matthews
authors.forename: E A
authors.initials: EA
authors.lastname: Wong
authors.forename: P K
authors.initials: PK
authors.lastname: Bender
authors.forename: K E
authors.initials: KE
authors.lastname: Webb
mesh_terms: D000818:Animals; D001483:Base Sequence; D016373:DNA, Antisense / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D005260:Female; D007408:Intestinal Absorption / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007583:Jejunum / Q000378:metabolism* / Q000502:physiology; D007930:Leucine / Q000493:pharmacokinetics*; D008239:Lysine / Q000493:pharmacokinetics*; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D013552:Swine / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D016373:DNA, Antisense; D012333:RNA, Messenger; D007930:Leucine; D008239:Lysine