Subcellular distribution of rat brain cortex high-affinity, sodium-dependent, glycine transport sites.
The subcellular distribution of the membrane components, present in rat brain cortex homogenates, that interact with glycine in the presence of sodium ions was studied. The distribution in the primary fractions, as per cent of total binding in the homogenate, was: P1 ('nuclear'), 58%; P2 (large granule), 39%; P3 (microsomal), 2%9 Of the subfractions obtained by centrifuging P1 in a linear 0.32--1.5 M sucrose gradient, only the lighter fraction (P1-III) formed by large myelin fragments was enriched in specific binding activity with respect to P1. The pellet formed by purified nuclei had negligible binding, and fractions of intermediate density had a lower activity than P1. Transient exposure of P1-III to 1.5 M sucrose did not diminish its binding ability. Similarly, in the subfractions obtained by centrifuging P1 in a discontinuous sucrose gradient, only the least dense one, P1-A, that is formed exclusively by large myelin fragments, was enriched with respect to P1. The electron microscopy of these fractions is presented. The P2 subfractions, obtained in a linear 2--18% Ficoll gradient, had the following sodium-dependent activity (counts/min/mg protein, fractions being in the order of decreasing density): pellet, 0; P2-I, O; P2-II, 450; P2-III, 1770; P2-IV, 4130; unfractionated P2, 880; P2-IV, the least dense fraction being composed mainly of myelin. With P2 subfractions obtained in a discontinuous sucrose gradient (0.32, 0.8 and 1.2 M sucrose layers), it was also found that sodium-dependent glycine binding was only enriched, with respect to P2, in the myelin fraction P2-A. Glycine binding to purified brain cortex myelin was also found to be very high, while binding to non-myelin membranes, obtained during the purification procedure, was only 0--7% of that seen with myelin. These results suggest that high-affinity glycine binding is located in myelin proper, and possibly also in some other glial plasma membranes, but not in nuclei, mitochondria, endoplasmic reticulum or synaptosomes. The relevance of these findings for interpreting previous reports on high-affinity glycine transport in the central nervous system is analyzed.
D000818:Animals; D001665:Binding Sites; D002540:Cerebral Cortex / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D009186:Myelin Sheath / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013347:Subcellular Fractions / Q000648:ultrastructure
D012964:Sodium; D005998:Glycine