Na +-dependent amino acid uptake by human placental microvillous membrane vesicles: Importance of storage conditions and preservation of cytoskeletal elements

Abstract

Human placental microvillous membrane vesicles (MMV) were purified by precipitation of non-microvillous membrane with MgCl 2 Two aspects of MIMV preparation were found to be important to the interpretation of amino acid transport studies: (1) storage conditions, and (2) preservation ofcytoskeletal elements. In non frozen MM V, MeAIB uptake was stimulated by an inward Na + gradient and showed ‘overshoot’. The initial Na + -dependent uptake rate was concentration-dependent with a V max of 640 ± 80 pmol/mg/30 sec and a K m 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°C or -196°C markedly reducedNa +-dependent uptake ofseveral amino acids, compared to vesicles stored at 4°C. The greatest reduction was seen with storage at -80°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 MM V.