The Apoproteins

Preliminary studies on the apoproteins of human and bovine erythrocyte Cu2Zn2-superoxide dismutase showed that the purest species are obtained using a gelfiltration column previously equilibrated with EDTA Other methods to remove the metal ions are dialysis against cyanide or diethyldithiocarbamat There are 

The enzymatic activity of the zinc-free protein is pH-dependent whereas native CUjZnjSOD is active over a wide pH-range (pH 5.0-9.5) Cu(II) in the zinc binding site gives little superoxide dismutase activity. This conclusion arises from the observation that AgjCUjSOD in which copper is in the zinc binding site, has almost no activity and that the Cu Cu enzyme has nearly the same activity as 

The apparent binding constant of copper to the apoprotein were investigated with equilibrium dialysis Four different constants are found. They are pH-dependent. At low pH, two protons compete with copper in the native binding sites. This is not seen at pH 7.0 and above. At alkaline pH-values (pH 10.0) the binding constants are nearly similar. Simultaneous addition of equimolar copper and zinc at pH 5.0 to the apo enzyme results in the formation of an electrophoretically distinct metal-deficient protein species and in the incorporation of copper and zinc in a one-to-one ratio at various concentrations of added metal ions At low ratios of added equimolar metals, not all of the metal ions expected are bound. This may account for the low yields often obtained in reconstitution studies. 

Recently the reaction of diethyldithiocarbamat (DDC) with Cu Zn superoxide dismutase was scrutinized The formation of an enzyme bound Cu(II) DDC complex was postulated after treatment of SOD with DDC. These results were challenged by a reexamination of the reaction of DDC with Cu2Zn2Superoxide dismutase A ternary Cu(II)-DDC-protein complex could not be detectal spectroscopically. The copper was completely removed from the protein. Hence, diethyldithiocarbamat is now as ever a good reactant for the preparation of the apoprotein of Cu2Zn2SOD. 

Another study is available on the metal replacement in iron superoxide dismutase from P. ovalis. The apoprotein was prepared by titration to alkaline pH-values The enzyme was reconstituted using Cr, Cd and Mn-ions All substituted SOD s had no enzymatic activity. The chiroptical properties, however, were similar to those 

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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. 

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). 

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

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-... 

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). 

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. 

R.J. Carrico and H.F. Deutsch, Presence of zinc in human cytocuprein and some properties of the apoprotein. J. Biol. Chem. 245, 723-727 (1970). 

Rare patients respond to the administration of thiamine in large doses (10-30mg/day). The clinical course is even more mild than that of patients with intermittent disease. Thiamine is a cofactor for the branched-chain ketoacid dehydrogenase, and the presumed mutation involves faulty binding of the apoprotein to this vitamin. 

Mitochondrial DNA is inherited maternally. What makes mitochondrial diseases particularly interesting from a genetic point of view is that the mitochondrion has its own DNA (mtDNA) and its own transcription and translation processes. The mtDNA encodes only 13 polypeptides nuclear DNA (nDNA) controls the synthesis of 90-95% of all mitochondrial proteins. All known mito-chondrially encoded polypeptides are located in the inner mitochondrial membrane as subunits of the respiratory chain complexes (Fig. 42-3), including seven subunits of complex I the apoprotein of cytochrome b the three larger subunits of cytochrome c oxidase, also termed complex IV and two subunits of ATPase, also termed complex V. 

Atxl chaperone apoprotein (without copper) shows tertiary structural differences in the loop area containing the cysteine ligand residues. Structural data for the apoprotein are deposited with PDB code 1FES.116... 

Among the relevant points to emerge from these studies is that yes, the structural differences between the oxidized and reduced forms of the blue copper proteins are rather small, but the structures are also quite similar to those of the apoproteins. Thus, the presence of the copper ion, in either oxidation state, does not impose any significant strain on the peptide chain. The amount of strain in the metal-ligand bonds has been debated, but it is clearly not a huge quantity in view of the fact that the affinities of the protein for the metal ions are quite high. 

Flavin Coenzymes.—5-Deazaflavin-adenine dinucleotide (2) can be prepared from the 5-deazaFMN,21 using a FAD pyrophosphorylase from rat liver.22 When the apoprotein of D-amino-acid oxidase from pig kidney is reconstituted with (2), no oxidation of D-alanine is observed, although the flavin chromophore in the reconstituted enzyme is reduced on the addition of DL-amino-acids.22 This has been interpreted as indicating that hydrogen transfer from the amino-acid to (2) can still... 

A few years later, in 1953, the versatility of pyridoxal phosphate was illustrated by Snell and his collaborators who found many of the enzyme reactions in which pyridoxal phosphate is a coenzyme could be catalyzed non-enzymically if the substrates were gently heated with pyridoxal phosphate (or free pydridoxal) in the presence of di- or tri-valent metal ions, including Cu2+, Fe3+, and Al3+. Most transaminases however are not metal proteins and a rather different complex is formed in the presence of the apoprotein. 

Lipoproteins (Table 5.2) are macromolecular aggregates with varying proportions of triglycerides and cholesterol (with some phosphoacylglycerols) and apoproteins. The apoproteins act as recognition flags for receptor binding, for example apo B and apo E,... 

In 1971, adrenodoxin, an iron-sulfur protein with a single tyrosine residue and no tryptophan was shown to fluoresce at 331 nm upon 280-nm excitation at neutral pH/20 1 On cooling from room temperature to 77 K, the emission maximum shifts to 315 nm. The redox state of the iron does not have any effect on the tyrosine emission. From these results, an exciplex between the excited singlet state of tyrosine and an unidentified group was suggested as the cause of the anomalous emission energy/2031 Later studies have shown that the excitation spectrum is a red-shifted tyrosine spectrum, that removal of the iron to form the apoprotein has no effect on the emission, and that heat, low pH, guanidine hydrochloride, urea, and LiCl all cause the emission... 

The gadolinium complex GdHPDOSA can be trapped inside the interior of apoferritin (60). The process is based on the dissociation of the apoprotein into subunits at low pH in a concentrated solution of the complex followed by its reforming at pH 7. The complex molecules that are not trapped inside the protein are eliminated by dialysis. The longitudinal relaxivity of the... 

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