8.3.4—
Golgi Membranes

Membrane fractions enriched in Golgi bodies are prepared by density-gradient centrifugation of organelles obtained after chopping plant tissues. Glutaraldehyde is routinely added to the extraction medium to 'stabilize' the structure of the Golgi bodies though it now seems that the organelle survives preparation intact and only requires this fixative for electron microscopy (Bowles & Kauss, 1976). Golgi bodies from plant sources sediment with the fraction of buoyant density 1.12 to 1.15 g cm^–3 in sucrose gradients and are identified in gradient fractions by their distinctive morphology. Thin section studies of whole cells in higher plants show the Golgi body as a stack of 5 to 8 closed membrane sacks. Each sack, or 'cisterna', is flattened to a disc, 0.5 to 1 m m in diameter. Around the perimeter of the disc there is progressive fenestration through, and localized swelling of, the cisternal membranes, grading into isolated vesicles beyond the edge of the disc (Fig. 8.4). In some cases a single layer of parallel fibrils has been seen in the 10–15 nm wide, electronlucent region between the cisternae. Freeze-etch pictures (Fineran, 1973a) confirm this structure for the organelle. Electron-microscope sections and negative-stain images of glutaraldehyde-fixed material show that this structure is maintained in isolated organelles. The cisternae remain joined in a stack as they are in the cell, but separate in media of high ionic strength (Mollenhauer et al., 1973).

Inosine diphosphatase (IDPase) has been used as a marker for Golgi bodies and, for some plant tissues, a single peak of activity is observed on sucrose gradients, coinciding with the Golgi body-rich fraction. However, IDPase activity

Figure 8.4
The structure of the Golgi body. Based on the
freeze-etch pictures of Fineran (1973a) which
are probably close to the real shape of this
organelle in the cell. Conventional electron
microscope methods involving fixation and
dehydration are more likely to cause distortion.

was found in purified nuclear membrane (Philipp et al., 1976), and using mung bean hypocotyl a second peak of activity was observed in light membrane characterized as ER (Bowles & Kauss, 1976).

Thin sections of Golgi body fractions from sucrose gradients show that a variety of less organized vesicles is also present, and the possibility that these preparations are contaminated with other membrane types cannot be excluded. The purest Golgi body fractions to date have been obtained from rat liver. Giving a rat ethanol induces the liver Golgi bodies to fill up with light lipoprotein particles which cause a distinctive change in the buoyant density of the organelle and serve as internal structural markers for electron-microscopy. The purified Golgi body membranes contained the cytochrome b₅ electron-transport chain (NADH-specific cytochrome c reductase and cytochrome b₅ ) characteristic of ER, but lacked NADPH-specific cytochrome c reductase and some other ER markers. UDP galactose:N-acetyl-glucosamine galactosyltransferase was restricted to the Golgi body fractions (Bergeron et al., 1973). Other evidence from animal cells indicates that fucosyl and sialyl transferases are primarily associated with the Golgi apparatus. 'Pure' Golgi fractions have not yet been prepared from plant material, but it has been shown that maximum activities of a UDP galactose:N-acetyl-glucosamine galactosyltransferase involved in glycoprotein biosynthesis (Powell & Brew, 1974), and UDP glucose:sterol glucosyltransferase (Bowles et al., 1976) were found in the Golgi body-enriched fraction of sucrose density gradients. The evidence then, such as it is, suggests that Golgi membranes have some affinity with the ER, but are specially enriched in glycosyltransferases involved in the synthesis of glycoprotein and glycolipid.