Microsomes electron microscopy

We chose adult female Dlploptera punctata as the test organism because the effects of precocene II on this insect have been described in detail (30) and because the CA of this insect produce exclusively JH III. Precocene II Is bioactivated by a mono-oxygenase in D. punctata CA, just as precocene I Is bioactivated in Locusta mlgratorla CA (11, 12). Indirect evidence for precocene II bioactivation to a highly reactive 3,4-epoxide Is not only provided by electron microscopy which shows extensive damage to the CA cells after in vivo or in vitro treatment (30). In addition, in vitro studies show that radio labelled precocene II is metabolized by I). punctata CA to a mixture of els- and trans-3,4-dihydrodiols which can be extracted and analyzed by HPLC (Fig. 2, HPLC conditions ether/pentane 1/1 + 0.2% 2-propanol 1.5 ml/min. 300 x 4 mm Micropak Si 5). The ratio of precocene II cis- and trans-3,4-dihydrodiols (36 64) is remarkably similar to the ratio of dihydrodiols obtained In incubations of rat liver microsomes with precocene II (16) or In incubations of L. mlgratorla CA with precocene I (11). 

The microsomes, when examined by electron microscopy, appeared to be small, globular, or bottle-shaped, closed vesicles bounded by a single smooth-surfaced membrane. They had no cytochrome c oxidase activity, and they were judged to be free of mitochondrial contamination. They did have 5 -nucleotidase activity. All agreed that the microsomes were derived from the canalicular membrane and were the site of acid secretion. 

Investigations have been carried on 3 subcellular compartments microsomes (4), mitochondria (5) and cytosol (6) their purity has been checked by electron microscopy and marker enzymes. After incubation, the radioactive fatty acids biosynthesized are extracted, methylated and analysed by a combination of thin-layer and gas-liquid chromatography (7). 

The plasma membrane enriched fraction was prepared by discontinuous sucrose density gradient centrifugation of a microsomal fraction prepared from total root homogenate freed from mitochondria, plastids and nuclei by differential centrifugation. Except for minor modifications the isolation procedure was as described by Hodges et al. (1972). The plasma membrane fraction was identified by enzyme reactions and by electron microscopy with PACP staining. Total lipids were extracted from the suspended membranes... 

Figure 1. Morphological analysis of dog pancreatic rough ER microsomes. (A) Electron micrograph of dog pancreatic rough microsomes. Arrows indicate ribosomes at the surface of ER microsomal membranes. (B) Contact mode Atomic Force Microscopy micrograph of purified ER microsomes fused on mica. Scale bars are 1 jxm.

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