Cystine-binding protein

Cystine-binding protein assay [5]. This indirect but rapid measurement method has a very high sensitivity and specificity, but disadvantages are the use of radioactive material and the questionable availability of the cystine-binding protein. It does not provide internal control of the sample tested. 

Oshima RG, Willis RC, Furlong CE, Schneider JA. Binding assays for amino acids. The utilization of a cystine binding protein from Escherichia coli for the determination of acid-soluble cystine in small physiological samples. J Biol Chem 1974 249 6033-6039... 

Sulfur-containing amino acids such as cystine and homocystine tend to bind to plasma proteins. This binding is irreversible hence, these amino acids will be severely underestimated unless the plasma is deproteinized immediately following its separation from red cells. Blood should be left standing for as short a time as possible to avoid binding of cystine to proteins and hemolysis. 

One of the hallmarks of OBPs is the six cysteine (six half cystines) residues, but this criterion alone is not sufficient to classify a certain protein as olfactory protein. It is important to demonstrate that an OBP is expressed only (or predominantly) in olfactory tissues. Evidence for their ability to bind odorants is also desirable, but not sine qua non. One of these criteria alone would not be enough to define a given protein as an OBP. For example, bovine serum albumin (BSA) binds to insect pheromones (Leal, unpublished data) and yet it is not an OBP because it does not occur in olfactory tissues in the first place. Conversely, a protein specific to antennae is not necessarily an OBP. There are other proteins that may be expressed in antennae but not in control tissues. Non-OBPs specific to insect antennae have been previously detected (Ishida and Leal, unpublished data). Also, a g lu tath i o n e -. S -1 ra n s I e ra s e has been reported to be expressed specifically in antennae of M. sexta (Rogers et al., 1999). Likewise, the six-cysteine criterion should not be misleadingly used. Insulin and bovine pancreatic trypsin inhibitor, for example, have six cysteines in three disulfide bridges and yet they are not odorant-binding proteins. Also, mammalian and insect defensins have six well-conserved cysteine residues. 

Fig. 8. Cys2-His2 zinc finger DNA-binding proteins contain multiple tandem repeats of zinc finger domains. A ribbon representation of a six-zinc-finger protein (white) is wrapped around the major groove of DNA (black). A single domain (right) consists of two /3-strands and an ce-helix. A zinc atom (sphere) is coordinated by two cystines and two histidines. Sequence-specific contacts with the DNA occur at the A-terminus of the ce-helix (top right of domain shown at right).

Many similarities, and strong dissimilarities of the known enzymes have been mentioned above. They are all identical in that a nucleotide-binding protein, with redox-active cysteine/cystine residues, combines with another metal-containing polypeptide or a metal coenzyme in which radical intermediates can be generated and stabilized. The binding protein carries several nucleotide sites so that reaction rates are influenced by allosteric effects, with the same specificity pattern everywhere. On closer inspection it becomes apparent that the considerable individual differences in subunit composition and in the nature of the second, catalytic component can indeed be integrated into a general concept of ribonucleotide reduction. 

FIGURE 16 Ribbon representation of irregular structures (a) High potential iron-sulfur protein coordinated to a 4Fe-4S cluster, (b) RAG1 DMA binding protein which contains representative examples of Zn-tinger domains, (c) Defensin as example of a membrane toxin stabilized by disulfide bonds, and (d) Chinese bird spider neurotoxin which contains a cystine knot. 

The reaction that leads to BCA color formation as a result of the reduction of Cu2+ is also strongly influenced by the presence of any of four amino acid residues (tyrosine, tryptophan, cysteine, or cystine) in the amino acid sequence of the protein. Unlike the Coomassie dye-binding (Bradford) methods, which require a minimum mass of protein to be present for the dye to bi nd, the presence of only a single amino acid residue in the sample may result in the formation of a colored BC A-Cu+ chelate. This is true for any of the four amino acids cited above. Studies done with di- and tripeptides indicate that the total amount of color produced is greater than can be accounted for by the simple addition of the color produced with each BCA-reactive amino acid, so the peptide backbone must contribute to the reduction of copper as well. 

BMP-2 has in common with BMP-7 and other members of the TGF-P family, a structural scaffold, consisting of a cystine-knot motif and two finger-like double-stranded P-sheets which determine the mode of dimerization. Secondary-structure differences between BMP-2 and BMP-7 account for the recognition and specific interaction with different binding partners. The crystal structure of human bone morphogenetic protein-2 is shown in Fig. 6.2. 

Platinum compounds have been widely used. These include the PtCl ion or the less reactive cis or trans platinum diaminodichloride compounds. At acid pH they react with methionine, cystine disulphides, N-termini or histidine. In the presence of ammonium sulphate, the chloride ions are rapidly substituted by ammonium ions to form [Pt(NH3)4] which is unreactive. Square planar negatively charged complexes such as Pt(CN)4 have been found to be effective in binding at the nucleotide-binding site in dehydrogenases. The cyanide ligands are firmly bound to the metal and are not displaced by protein atoms. 

The thermal stability of thermolysin, a peptidase from the thermo-phillic bacterium Bacillus thermoproteolyticus, is apparently conferred by calcium bound to the enzyme (194), which has a molecular weight of 35,000 (195), and binds one mole of zinc (195) and three moles of calcium per mole of protein (196). Thermolysin contains no non-protein constituents (197) and consists of a single peptide chain containing neither cysteine nor cystine (197). Thermolysin has a high content of aspartic... 

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