Brain transferrin receptors and the distribution of cytochrome oxidase.

aspirated and the colloid was removed by washing five times in an incubation medium containing 150 mM-NaC1, 5 mMKCI, 15 mM-Mops (pH 7.5 with KOH) and 5 mM-glucose. Cell suspensions for transport assays were confirmed as 1 OO'% reticulocytes as judged by supravital staining with Brilliant Cresyl Blue [ 31. IT-Labelled amino acid influx was measured by the oil-stop methodology described in detail by Young & Ellory [4]. Fig. 1 shows the uptake of ~[U '~C]a lan ine (0.2 mM extracellular concentration, 37°C) by reticulocytes from an anaemic Tr'-type sheep measured in the presence and in the absence o f Na'. The results demonstrate the presence of Na + -dependent L-alanine transport activity in sheep reticulocytcs and show that incubation times of 1 min may bc used to measure initial rates o f i.-alanine uptake. To characterize further L-alaninc transport in sheep reticulocytes (from a Tr--type animal), we measured the initial rate of L[U'JC]alanine uptake ( 1 ) ) as a function of extracellular Lahnine concentration ( s ) over the range 0.05-5.0 mM in NaCl and KCI media at 37°C. Na'-dependent L-alanine transport in shccp reticulocytes was saturablc. Kinetic constants estimated from a plot of s / ~ ) versus s were: V,,,,, = 22.5 mmol/h per litre o f cells; apparent K, , = 0.Y mM. Evidence that Na' -dependent L-alanine transport in shccp rcticulocytcs is mediated by system ASC [S) was procompetition experiments. Effects of 1 0 mML-cysteine, L-serine, [.-leucine, L-valine, glycine, D-alaninc, L-lysine, and L-arginine on Na + -dependent I.-[ U'Tlalaninc uptake ( I mM extracellular concentration, 37°C) (expressed as percentage inhibition) were: 100, 89, 40, 30, 25 , 20, 0 and 0, respectively, for Tr'-type cells (control = 5.48 mmol/h per litre of cells); and 95, 88, 57. 45, 30, 30, 16 and 15% in reticulocytes from a Tr--type animal (control = 1 I . I mmol/h per litre of cells). In contrast t o these results, Na+-independent L-alanine transport was most effectively inhibited by [.-leucine. For Tr + -type reticulocytes in KCI medium, the concentration of L-leucine required to cause half-maximal inhibition of 1 mM[.-(UIJC]alanine uptake as <0.25 mM. We interpret this observation as indicative o f the presence of a high-affinity system L variant in sheep rcticulocytes. Interestingly, a significant proportion o f the Na'-independent L-alanine flux was insensitive to L-leucine at concentrations of 1 0 mM or more. This residual component of transport was approximately 1 mmol/h per litre of cells in Tr+-type reticulocytes and 0.56 mmol/h per litre of cells in Tr--type reticulocytes ( 1 m ~ ~ [ U ~ ~ C ] a l a n i n e d 37°C). These transport rates are of the same order as that found in mature sheep erythrocytes [ 1 , 21. We therefore attribute this Na+-independent Lleucine-insensitive L-alanine uptake route to transport by system asc(C'). In conclusion, the present results suggest that Na+dependent (system ASC) and Na' -independent [system u.sc(C] 1 1.-alanine transport routes co-exist in Tr'-type and Tr ~ -type sheep reticulocytes. Expression of the transport 0.5