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.
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.
Matthews
J C
JC
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306, USA.
Wong
E A
EA
Bender
P K
PK
Webb
K E
KE
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
D016373:DNA, Antisense; D012333:RNA, Messenger; D007930:Leucine; D008239:Lysine