Essential amino acid splanchnic bed exchanges in the rat: effects of pregnancy and food deprivation

Abstract

approx. 45 000 described by Koepsell et al. (1984) was not present in any appreciable quantity in our preparation. In fact this protein was selectively removed at an earlier stage in the preparation of the proteoliposomes. We therefore suggest that, at least in rabbit renal brush-borders, the protein with M , of approx. 45000 plays no role in the Na' -dependent transport of alanine. All four bands associated with the proteoliposomes were glycoproteins. These results also suggest that the addition of a specific lipid is not necessary for transport, since we used a crude mixture. Koepsell et al. (1984) observed an increased uptake of alanine when their liposomes were formed from pure phosphatidylserine and cholesterol. It is possible that this resulted from the production of less leaky liposomes rather than from an enhancement of activity. As a possible method for producing less permeable liposomes, the number of freeze/thaw cycles before assaying was increased. Two cycles increased the Na' -dependent peak and reduced the rate of the Naf -independent 'leak'. A third cycle further reduced the leak but also diminished the Na+ -dependent peak. Hence it appears that two freeze/thaw cycles give the best results. As well as displaying a Naf -dependent alaninetransport activity, the proteoliposomes also possessed a Na+-dependent D-glucose-transport activity, showing that at least one other Na+ -dependent transport protein had been reconstituted along with the alanine transporter. Future work will be aimed at further purification of this latter protein so that it can be identified.