Apoprotein

Usually the apoprotein was prepared by employing excessive dialysis against cyanide, EDTA or 1,10-phenanthroline (69, 70, 71, 72). However, these apoproteins still contained considerable amounts of copper and zinc (5—20% of the original content). We have devised a new method (76, 78) using chelator-equilibrated gel columns. EDTA proved most convenient, as already observed (69, 72). Concentrated erythrocuprein samples were layered on top of a Sephadex-G.-25 column which was previously equilibrated with 10 mM EDTA, pH 3.8. The migration rate of the protein was adjusted in a way such that 8—10 hours elapsed before 

The reconstitution of the apoprotein with Cu2+ and Zn2+ was most successful under anaerobic conditions using the two-column technique (78). The first column contained the chelator-equilibrated gel it was connected to a second column as soon as the apoprotein started to appear. The upper 20 mm of the second G-25-Sephadex column was previously equilibrated with ImM Cu2+ and Zn2+. The transfer of the apoprotein to the second column was monitored at 253 nm and the two columns were disconnected when it was complete. The elution of the second column was continued using 5 mM potassium phosphate buffer, p H 7.2, previously saturated with N2. 

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Sacchettini, J.C., et al. Refined apoprotein stmcture of rat intestinal fatty acid binding protein produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 86 7736-7740, 1989. 

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

Kulinski, T., Visser, A. J. W. G., O Kane, D. J., and Lee, J. (1987). Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi. Biochemistry 26 540-549. 

Shimomura, O., and Shimomura, A. (1981). Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein. Biochem. J. 199 825-828. 

The Rieske protein in mitochondrial bci complexes is assembled when the protein is incorporated into the complex. The Rieske protein is encoded in the nucleus and synthesized in the cytosol with a mitochondrial targeting presequence, which is required to direct the apoprotein to the mitochondrial matrix. The C-terminus is then targeted back to the outside of the inner mitochondrial membrane where the Rieske cluster is assembled. In addition, the presequence is removed and the protein is processed to its mature size after the protein is inserted into the bci complex. In mammals, the presequence is cleaved in a single step by the core proteins 1 and 2, which are related to the general mitochondrial matrix processing protease (MPP) a and (3 subunits the bovine heart presequence is retained as a 8.0 kDa subunit of the complex (42, 107). In Saccharomyces cerevis-iae, processing occurs in two steps Initially, the yeast MPP removes 22 amino acid residues to convert the precursor to the intermediate form, and then the mitochondrial intermediate protease (MIP) removes 8 residues after the intermediate form is in the bci complex (47). Cleavage by MIP is independent of the assembly of the Rieske cluster Conversion of the intermediate to the mature form was observed in a yeast mutant that did not assemble any Rieske cluster (35). However, in most mutants where the assembly of the Rieske cluster is prevented, the amount of Rieske protein is drastically reduced, most likely because of instability (35, 44). 

Different metals are readily incorporated into rubredoxin-type centers after reconstitution of apoprotein with appropriate metal salts (5). Rd and Dx derivatives containing Ni and Co have been analyzed by UV-vis, NMR, EPR, electrochemistry, and MCD (13, 19-22). Fe replacement has been used for Mossbauer studies and an indium de-... 

Finally, a disagreement was noted between the iron determination results, which showed the existence of only 5 to 8 iron atoms/mole-cule, and the above analysis. As a solution to this discrepancy, it was proposed that the samples could contain a mixture of holo- and apoprotein. 

Oxygenation of Hemoglobin Triggers Conformational Changes in the Apoprotein... 

Figure 12-6. Iron-sulfur-protein complex (Fe4S4). d acid-labile sulfur Pr,apoprotein Cys, cysteine residue. Some iron-sulfur proteins contain two iron atoms and two sulfur atoms (FejSj).

The nonpolar lipid core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules (Figure 25-1). These are oriented so that their polar groups face outward to the aqueous medium, as in the cell membrane (Chapter 14). The protein moiety of a lipoprotein is known as an apo-lipoprotein or apoprotein, constituting nearly 70% of some HDL and as litde as 1% of chylomicrons. Some apolipoproteins are integral and cannot be removed, whereas others are free to transfer to other hpoproteins. 

LDL (apo B-lOO, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-lOO binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl ester are then hydrolyzed in the lysosomes, and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits in a coordinated manner HMG-CoA synthase, HMG-CoA reductase, and, therefore, cholesterol synthesis stimulates ACAT activ-... 

As an example, the low-density lipoprotein (LDL) molecule and its receptor (Chapter 25) are internalized by means of coated pits containing the LDL receptor. These endocytotic vesicles containing LDL and its receptor fuse to lysosomes in the cell. The receptor is released and recycled back to the cell surface membrane, but the apoprotein of LDL is degraded and the choles-teryl esters metabolized. Synthesis of the LDL receptor is regulated by secondary or tertiary consequences of pinocytosis, eg, by metabolic products—such as choles-... 

The phycobiliproteins are accessory photosynthetic pigments aggregated in cells as phycobilisomes that are attached to the thylakoid membrane of the chloroplast. The red phycobiliproteins (phycoerythrin) and the blue phycobiliprotein (phycocy-anin) are soluble in water and can serve as natural colorants in foods, cosmetics, and pharmaceuticals. Chemically, the phycobiliproteins are built from chro-mophores — bilins — that are open-chain tetrapyrroles covalently linked via thio-ether bonds to an apoprotein. ... 

The algal extract of P. aerugineum is blue, with maximum absorbance at a wavelength of 620 nm and a red fluorescence with maximum emission at 642 nm. The main phycobiliprotein, C-phycocyanin, is the same type of phycocyanin found in most Cyanobacteria. The chromophores are composed of phycocyanobilins, conjugated to an apoprotein via thioether bonds. 

Extensive studies in vitro from many groups have confirmed that exposure of LDL to a variety of pro-oxidant systems, both cell-free and cell-mediated, results in the formation of lipid hydroperoxides and peroxidation products, fragmentation of apoprotein Bioo, hydrolysis of phospholipids, oxidation of cholesterol and cholesterylesters, formation of oxysterols, preceded by consumption of a-tocopherol and accompanied by consumption of 8-carotene, the minor carotenoids and 7-tocopherol. 

FIGURE 5.1 Rapid molecular simulations of the apoprotein form of a-lactalbumin in vacuo, showing the native holo state and the effect of simulations at 5 and 298 K of the apoform (Farrell et ai, 2002). 

Baer, B. R. Wienkers, L. C. Rock, D. A. Time-dependent inactivation of P450 3A4 by raloxifene identification of Cys239 as the site of apoprotein alkylation. Chem. Res. Toxicol. 2007, 20, 954-964. 

Aro, A., Teirila, J., Gref, C-G., Dose-dependent effect on serum cholesterol and apoprotein B concentrations by consumption of boiled, non-filtered coffee, Atherosclerosis, 83, 257, 1990... 

Becker, N., Illingsworth, R., Alaupovic, P., Connor, W. E., Sundberg, E. E., Effects of saturated, monounsaturated, and w-6 polyunsaturated fatty acids on plasma lipids, lipoproteins and apoproteins in humans, Am J Clin Nutr, 37, 355, 1983... 

Lipoproteins. A lipoprotein is an endogenous macromolecule consisting of an inner apolar core of cholesteryl esters and triglycerides surrounded by a monolayer of phospholipid embedded with cholesterol and apoproteins. The functions of lipoproteins are to transport lipids and to mediate lipid metabolism. There are four main types of lipoproteins (classified based on their flotation rates in salt solutions) chylomicrons, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). These differ in size, molecular weight, and density and have different lipid, protein, and apoprotein compositions (Table 11). The apoproteins are important determinants in the metabolism of lipoproteins—they serve as ligands for lipoprotein receptors and as mediators in lipoproteins interconversion by enzymes. 

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