Specific peptides

Different substances, such as lipids, lipoproteins, hormones, fractions of plasma, toxins, etc., are included in the last part of Table 4.7.5. From the point of view of practical application of bioaffinity chromatography an important example is industrial blood plasma fractionation [220]. 

2 Jacoby, W.B. and Wilchek, M. (eds.) (1974) Methods in Enzymology, Vol. 22, Enzyme Purification -Part B, Affinity Techniques, Academic Press, New York. 

3 Lowe, C.R. and Dean, P.D.G. (1974) Affinity Chromatography, John Wiley and Sons. London. 

4 Ruoslahti, E. (ed.) (1976) Immunoadsorbents in Protein Purification, University Park Press, Baltimore. 

6 Epton, R. (ed.) (1978) Chromatography of Synthetic and Biological Polymers, Vols. 1 and 2, Ellis Horwood, Chieester. 

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Selective hydrolysis can be accomplished by using enzymes to catalyze cleavage at specific peptide bonds... 

INSULIN. Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin, an important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin (Figure 15.3). 

There have been notable successes in the replacement of individual peptide residues by peptoid monomers with retention of in vitro activity and enhancement of specificity. Unfortunately, attempts to completely transform those bioactive peptides that function via specific peptide-protein binding events into entirely pep-toid-based ohgomers have so far proven successful only at short chain lengths (e.g. [23]). It remains to be seen whether any general strategy can be developed in... 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

The insulin-like growth factor I receptor is closely related to the insulin receptor. The RTK activity of the IGF-I receptor is regulated by intermolecular autophosphorylation at three sites within the activation loop. The crystal structure of the trisphosphorylated form of IGF-I RTK domain with an ATP analog and a specific peptide substrate showed that autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Furthermore, the structure revealed how... 

His residues in the peptide sequence are fundamental for Ni11 ion interactions with peptides. When the His residue is relatively distant from the N terminus, it may compete as a primary ligation site with the N-terminal amino nitrogen. However, even the higher number of His residues inserted inside the peptide sequence may be not able to compete with the albumin-like N terminus, unless the specific peptide structure is established. 

Figure 12.4 MALDI analysis of peptides prepared in situ from a 10 1 mixture of Bacillus cereus and Bacillus anthracis sp. Sterne.82 (a) Survey spectrum of peptide products. Species assignments are indicated on the figure. (b) Spectrum of fragment ions produced by low-energy collisions of the Bacillus cereus-specific peptide of mass 1529. (c) Spectrum of fragment ions produced by low-energy collisions of ions of the Bacillus anthracis peptide of mass 1528.

Alternatively, species-specific peptides can be targeted and partially sequenced to confirm the presence or absence of microorganisms of high interest. 

The synthesis and characterization of a somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, labeled with an indo-dicarbo- and an indotricarbocyanine dye at the V-terminal amino group were described in [34], The ability of these fluorescent contrast agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, wherein the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. The intracellular localization was visualized using NIR fluorescence microscopy. 

The immobilized immunoprecipitates are washed twice with lysis buffer containing 0.5 MNaCl and twice with buffer A. The beads are resuspended in 20 /il of kinase buffer also containing the appropriate concentration of the specific peptide. Reactions should also be set up without peptide as a negative control for nonspecific or self-incorporation of radiolabel. To start the reactions, 5 /il of ATP is added (final concentration 0.1 mM unlabeled ATP, 1 /iCi [7 -32P]ATP (per assay) in kinase buffer). The assays are allowed to proceed for 15 to 30 min at 30° with constant shaking at 900 rpm, and stopped by spotting 20 /il of the sample (slurry) onto a square (1.5 X 1.5 cm) of phosphocellulose (P81) paper. The P81 papers are immediately immersed in 500 ml of 1% (v/v) orthophosphoric acid, and then washed 3 times with the same solution (to remove the excess ATP). The washes therefore contain almost all of the radiolabel and must be handled carefully and disposed of appropriately. The papers are briefly rinsed in ethanol and air-dried. The incorporation of 32P-label is measured by Cerenkov counting. 

There are two types of specificity checks that may be warranted when choosing a specific peptide. The first is to demonstrate that the peptide is bound by the desired antibody and not by other, antigenically irrelevant antibodies. An example of this kind of specificity check is shown in Figure 7.3. A peptide that is immunoreactive with the 1D5 estrogen receptor (ER) mAb was covalently bound as a 1 pL spot to the center of each grid location. Various antibodies and controls were subsequently applied to the different grid locations. The bottom panel describes each of the antibodies that were applied to each grid location. The ability of the various antibodies to bind to the peptide was tested by immunohistochemistry. The presence of antibody bound to a... 

Column A (Fig. 16.5) shows the baseline binding capability of each antibody for its peptide epitope. Prior experiments had established that each antibody only binds to its specific peptide and not the others.15 The presence of a colored spot on the glass slide indicates immunoreactivity. Each row contains a different peptide and antibody combination, as indicated by the legend to the left. The legend at the top of Figure 16.5 indicates that the peptides in column A are not treated in any way. 

This technique has been described as a general method of studying protein-protein interactions as well as a method for investigating the three-dimensional structure of individual proteins (Muller et al., 2001 Back et al., 2003 Dihazi and Sinz, 2003 Sinz, 2003 Sinz, 2006). It also has been used for the study of the interactions of cytochrome C and ribonuclease A (Pearson et al., 2002), to investigate the interaction of calmodulin with a specific peptide binder (Kalkhof et al., 2005a Schmidt et al., 2005), and for probing laminin self-interaction (Kalkhof et al., 2005b). 

Utilization of cell-specific peptide sequences in biomaterials enables the selective adhesion of certain cell types, even in the presence of a mixture of many cell types. As mentioned earlier, REDV promotes the adhesion of endothelial cells, but not other vascular cell types (Hubbell et al., 1991). This selectivity has great potential for endothelialization of vascular devices, where the growth of an endothelial cells, but not fibroblasts or smooth muscle cells, is desired. Another peptide sequence, KRSR, has been shown to selectively promote the adhesion of osteoblasts, which is useful in the rational design of better dental and orthopedic biomaterials (Dee et al., 1998). 

Such modifications can be very helpful when the fragmentation efficiency of a peptide is poor or the number of sequence-specific peptides is limited. 

The most abundant milk protein is casein, of which there are several different kinds, usually designated a-, (1-, and K-casein. The different caseins relate to small differences in their amino acid sequences. Casein micelles in milk have diameters less than 300 nm. Disruption of the casein micelles occurs during the preparation of cheese. Lactic acid increases the acidity of the milk until the micelles crosslink and a curd develops. The liquid portion, known as whey, containing water, lactose and some protein, is removed. Addition of the enzyme rennet (chymosin) speeds up the process by hydrolysing a specific peptide bond in K-casein. This opens up the casein and encourages further cross-linking. 

Peptidergic nerves are known to innervate tissues with a high mast-cell content and to contain specific peptides which have been demonstrated to affect mast-cell function [5, 54-56]. 

Another aspect of stimulus-secretion coupling in the mast cell in which specific peptides and compound 48/80 have been studied is the phosphorylation of specific protein bands in response to stimulation [211-214], In these experiments mast cells were pre-labelled with 32P and stimulated, and... 

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