Stabilization and Labilization of Eysosomes in Rat Retina

acetylneuraminic acid hydrolase the kidney cortex homogenate was subjected to differential centrifugation (see Table 1). Each subcellular fraction obtained was washed twice by repeated centrifugation and the pooled supernatants were then subjected to the next centrifugation step. In this way the homogenate was separated into five particulate fractions and one soluble fraction. The subcellular fractions were assayed for CMP-Nacetylneuraminic acid hydrolase, protein. and various membrane marker enzymes, such as alkaline phosphatase (brush border), ouabaine-sensitive (Na+-K+)-ATPase (plasma membranes), thiamine pyrophosphatase (Golgi membranes), a2-acid glycoproteinsialyltransferase (Golgi membranes) and glucose 6-phosphatase (endoplasmic reticulum membranes) [for assay methods see Van Dijk et al. (1973), except for sialyltransferase (W. Van Dijk & D. H. Van den Eijnden, unpublished work)]. From the results given in Table 1 it appears that CMP-N-acetylneuraminic acid hydrolase is predominantly present in the light and heavy microsomal fractions (39 and 2079, whereas the heavy mitochondrial fraction also contains a considerable activity (21 %). However, only the microsoma1 fractions show a higher specific activity than the homogenate (3.1 and 2.1). The other particulate fractions show equal or lower specific activities than the homogenate. No enzyme activity was detectable in the soluble fraction. When the results for CMP-N-acetylneuraminic acid hydrolase activity are compared with the results for the membrane marker enzymes, it appears that only (Na+-K+)ATPase shows a completely different distribution pattern, whereas the subcellular distribution of the other marker enzymes are more or less comparable with the CMP-Nacetylneuraminic acid hydrolase distribution. These results suggest that if (Na+-K+)-ATPase is a true plasma membrane marker, it is unlikely that CMP-N-acetylneuraminic acid hydrolase is located predominantly in plasma membranes, which is in contrast with the suggested localization by Kean and co-workers (Kean, 1972; Kean & Bighouse, 1973). However, from these results it is not possible to state unequivocally in which microsomal membranes CMP-N-acetylneuramink acid hydrolase is located: further subcellular studies are needed.