Na(+)-dependent amino acid uptake by human placental microvillous membrane vesicles: importance of storage conditions and preservation of cytoskeletal elements.
Human placental microvillous membrane vesicles (MMV) were purified by precipitation of non-microvillous membrane with MgCl2. Two aspects of MMV preparation were found to be important to the interpretation of amino acid transport studies: (1) storage conditions, and (2) preservation of cytoskeletal elements. In non-frozen MMV, MeAIB uptake was stimulated by an inward Na+ gradient and showed 'overshoot'. The initial Na(+)-dependent uptake rate was concentration-dependent with a Vmax of 640 +/- 80 pmol/mg/30 sec and a Km of 0.44 +/- 0.77 mM. Na(+)-stimulated cysteine uptake (65 +/- 23 pmol/mg/30 sec), previously thought to be very low or absent in the human placenta, was comparable to MeAIB, although there was no 'overshoot'. Cysteine uptake was partially stimulated by Li+. In general, freezing and storage at either -80 degrees C or -196 degrees C markedly reduced Na(+)-dependent uptake of several amino acids, compared to vesicles stored at 4 degrees C. The greatest reduction was seen with storage at -80 degrees C, especially with cysteine. There was no effect of storage temperature on Na(+)-independent amino acid uptake. For frozen vesicles, there was no difference in uptake for 12 versus 60 h storage. Removal of cytoskeletal proteins with the chaotropic agent, KSCN, resulted in greater enrichment of MMV marker proteins, but the preparation lost the capacity for active MeAIB uptake. These data, especially with regard to storage conditions, highlight the importance of precise definition of preparation and storage conditions when interpreting results of amino acid uptake by human placental MMV.
Karl
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PI
Department of Pediatrics, North Shore University Hospital, Cornell University Medical College, Manhasset, New York.
Teichberg
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S
Fisher
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SE
D000596:Amino Acids / Q000493:pharmacokinetics*; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000704:Analysis of Variance; D015415:Biomarkers; D002824:Chorionic Villi / Q000502:physiology*; D003545:Cysteine / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004082:Dihydroalprenolol / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis; D005615:Freezing; D005966:Glucuronidase / Q000096:biosynthesis; D006639:Histidine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis; D009708:Nucleotidases / Q000096:biosynthesis; D012694:Serine / Q000378:metabolism; D012964:Sodium / Q000502:physiology*; D013997:Time Factors
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D015415:Biomarkers; D003598:Cytoskeletal Proteins; C017911:2-(methylamino)isobutyric acid; D012694:Serine; D006639:Histidine; D004082:Dihydroalprenolol; D012964:Sodium; D009251:NADPH-Ferrihemoprotein Reductase; D003576:Electron Transport Complex IV; D009708:Nucleotidases; C030287:nucleotidase; D005966:Glucuronidase; D003545:Cysteine