Endoplasmic reticulum preparation

Both eukaryotes and prokaryotes have cell membranes that enclose the cell contents and act as barriers to the entry of a number of substances. Eukaryotes have only subcellular particles, including the nucleus. Microsomes are not present in either cell type. They are artifacts of endoplasmic reticulum preparation. 

The major mono oxygenases in the endoplasmic reticulum are cytochrome P450s—so named because the enzyme was discovered when it was noted that preparations of microsomes that had been chemically reduced and then exposed to carbon monoxide exhibited a distinct peak at 450 nm. Among reasons that this enzyme is important is the fact that approximately 50% of the drugs humans ingest are metabolized by isoforms of cytochrome P450 these enzymes also act on various carcinogens and pollutants. 

Electron microscopic examination of midge microsomes prepared by a slightly different procedure than Table VIII revealed a homogeneous mixture of vesicles derived from rough and smooth endoplasmic reticulum, ribosomes and a few mitochondria. Midge preparations are similar in composition to microsomal fractions of southern armyworm fat body and gut (44). 

Flavanone 3 -hydroxylase (F3 H ECl.14.13.21 CYP75B) activity was initially identified in microsomal preparations of golden weed (Haplopappus gracilis) [110]. E3 H from irradiated parsley cell cultures was later biochemically analyzed and characterized as a cytochrome P450 having an absolute requirement for NADPH and molecular oxygen as cofactors [111]. The enzyme has been shown to have activity with flavanones, flavones, dihydroflavonols, and flavonols, but does not appear to have activity with anthocyanidins [111]. The first cDNA clone for E3 H was isolated from Petunia [112]. It has been suggested that E3 H may serve as an anchor for the proposed flavonoid multi-enzyme complex on the cytosolic surface of the endoplasmic reticulum [44]. 

Fig. 2 Routine preparation of a hydrogenosome (H) in Trichomonas vaginalis (a), Tri-trichomonas foetus (b), and Monocercomonas sp. (c). Note that in T. foetus the hydrogenosome is spherical, enveloped by a double membrane (arrows), and presents a single peripheral vesicle, whereas in T vaginalis several peripheral vesicles are seen surrounding the organelle (arrows) and in Monocercomonas the hydrogenosome is very elongated. ER, endoplasmic reticulum N, nucleus. Bars = 100 nm. (From Benchimol 2001 Diniz and Benchimol 1998)...

Figure 8-26 The structure of the 994-residue Ca2+-ATPase of the endoplasmic reticulum of rabbit muscle at 0.8-nm resolution. (A) Predicted topology diagram organized to correspond to the electron density map prepared by electron crystallography of frozen-hydrated tubular crystals. The number of amino acid residues in each connecting loop is marked. (B) The electron density map with the predicted structure embedded. The relationships of the helices in (B) to those in (A) are not unambiguous. The helices marked B, D, E, and F in (B) may form the Ca2+ channel. The large cytoplasmic loops, which are black in (A), were not fitted. From Zhang et al.553 Courtesy of David L. Stokes.

Ribosomes were discovered by electron micros-copists examining the structure of the endoplasmic reticulum using ultrathin sectioning techniques. Their presence in cells was established by 1956, and the name ribosome was proposed in 1957. At first it was difficult to study protein synthesis in vitro using isolated ribosomes. No net synthesis could be detected until Hoagland and associates measured the rate of incorporation of 14C-labeled amino acids into protein.26 This sensitive method permitted measurement of very small amounts of protein synthesis in cell-free preparations from rat liver and paved the way toward studies with ribosomes themselves. 

In interesting recent studies, Poliak and co-workers (67, 68) have observed glucose-6-P phosphohydrolase, acid inorganic pyrophosphatase, and PPi-glucose phosphotransferase activities to be considerably higher in lipid-poor reticulosomes than in microsomal preparations. They hypothesized that reticulosomes, which they prepared by treatment of microsomal preparations from rat and chick livers with ribonuclease and deoxycholate (68), may be the precursors of endoplasmic reticulum, and... 

The P450 enzymes are found primarily in the outer membrane of the endoplasmic reticulum. Enzyme activity requires that the enzyme be integrated into a membrane that contains P450 reductase and, for some reactions, cytochrome b5. Characterization of the saturation kinetics for the P450 enzymes can be determined using a variety of enzyme preparations, including tissue slices, whole cells, microsomes, and reconstituted, purified enzymes. The more intact the in vitro preparation, the more it is likely that the environment of the enzyme will represent the in vivo environment. However, intact cell preparations do not... 

A number of important biochemical reactions occur intramitochondrially and some of these are described below. One major drawback to the study of energy metabolism in cestodes is the failure, due to technical difficulties, to produce isolated mitochondrial preparations of the purity which can be obtained from mammalian tissues. Generally, mitochondrial fractions of cestode origin are contaminated with other cellular components such as glycogen, endoplasmic reticulum and various membranes, and some of the data obtained with such mitochondria should, therefore, be treated with caution. 

Incubate a tissue slice at 37°C with glucose 6-phosphate in a suitable buffer solution. Wash the tissue free of the substrate, and then precipitate the phosphate ions by the addition of lead nitrate to the tissue slice. The remainder of the preparation is as described in Example 1.5. In liver cells the reaction product is found within the endoplasmic reticulum, thus indicating the location of the enzyme. 

Fragments of endoplasmic reticulum are transformed from lipid bilayer sheets, with attached ribosomes, into spherical vesicles. This is a result of the homogenization used in preparing the samples and also the tendency of lipid bilayers (Fig. 1-4) to spontaneously reseal. 

The original experiments on PDI activity were carried out with microsomal membranes, suggesting the localization of this protein in the endoplasmic reticulum. This has since been conhrmed both by subcellular localization studies (Lambert and Freedman, 1985) and by immunochemical methods (Koch, 1987). However, these studies revealed that PDI does not behave like a typical ER membrane protein. Indeed, for some time there was a question as to whether there was more than one form of PDI due to the presence of this protein in the cytosol. This has now been discounted by studies on the latency of PDI that show that if microsomes are prepared carefully then the activity is entirely latent (Lambert and Freedman, 1985). Thus, although PDI exists as an ER protein, it is easily released from microsomes by freeze thawing, mild alkaline treatment, detergents, or sonication, suggesting that PDI is either a soluble protein or is only loosely associated with the ER membrane. 

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