Isolation and Characterization of an Enriched Golgi Fraction from Neurons of Developing Rat Brains

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Abstract
We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10‐ to 12‐day‐old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem.31, 887–894 (1978)]. Golgi‐enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28–34% and 34–36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21–34% of fraction 1 and 11–29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6–10%) and fraction 2 (14–26%). Golgi fraction 1 showed a 25‐ to 65‐fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP‐sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8‐to 23‐fold enrichment over neurons of the activities of the above glycolipid‐ and glycoprotein‐synthesizing enzymes. The activities of the possible marker enzymes rotenone‐insensitive NADH‐cytochrome c reductase, succinate‐cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+,K+‐ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.

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