Proteins modifications. Table

We are attempting to understand the biological significance of the large variations in frequency of putative LDRs, whether between different types of bacteria or archaea, or between pro- and eukaryota. We have carefully studied the literature of more than 90 example proteins selected from our disordered protein databases and found reports on the functions of most of the disordered regions (Dunker et al., 2002). The observed functions and the number of examples in each functional class are given in Table VI. As indicated, four major functional classes were found molecular recognition, molecular assembly or disassembly, protein modification, and entropic chains. 

As discussed for N-myristoylation and S-prenylation, even S-acylation of proteins with a fatty acid which in the vast majority of cases is the C16 0 palmitic acid, plays a fundamental role in the cellular signal-transduction process (Table l). 2-5 14 While N-myristoylation and S-prenylation are permanent protein modifications due to the amide- and sulfide-type linkage, the thioester bond between palmitic acid and the peptide chain is rather labile and palmi-toylation is referred to as a dynamic modification. 64 This reversibility plays a crucial role in the modulation of protein functions since the presence or absence of a palmitoyl chain can determine the membrane localization of the protein and can also be used to regulate the interactions of these proteins with other proteins. Furthermore, a unique consensus sequence for protein palmitoylation has not been found, in contrast to the strict consensus sequences required for N-myristoylation and S-prenylation. Palmitoylation can occur at N- or C-terminal parts of the polypeptide chain depending on the protein family and often coexists with other types of lipidation (see Section 6.4.1.4). Given the diversity of protein sequences... 

Due to its high reactivity, NO can interact and react with many effector proteins. Targets are proteins with boimd metal ions and specific cysteine residues of proteins. In Table 6.3, some important bioregulatory proteins are summarized, for which direct modification by NO is assumed. Two target proteins should be mentioned in particular ... 

This part of the chapter will be restricted to FiiG-l-ASF because this type of modified 1-ASP seems to be the most promising for clinical studies. However, it should be mentioned that PEG modification is not standardized. The half-life varies depending on the molecular weight of PEG used as well as die degree of protein modification [31]. Table 4 describes the effect of PEGylation on half-lives in some animals. 

Among the more important factors affecting reactions with proteins, pH is the most important since it controls the distribution of potentially reactive side chains between reactive and unreactive ionization states (see Table II). Iodoacetic acid is a commonly used reagent in protein modifications and serves as an example. At low pH values (such as 2-5)... 

Table III. Possible Chemical Side Reactions during Protein Modification...

Table 5-6. Croup specific reagents for protein modification...

The oxidation of trypsin and trypsinogen was carried out in aqueous 0.1 M acetate buffer solutions at room temperature. In this particular case and under these conditions no significant cleavage of peptide bonds next to tryptophan residues occurred. Careful analysis of hydrolyzates of NBS-oxidized trypsinogen and trypsin confirmed the selectivity of the oxidative modification of the protein, as Table XXIV shows. There is no significant loss of tyrosine, histidine, serine, threonine, or cystine, although all of these amino acids will react with NBS but considerably less rapidly than tryptophan. 

Recently, a method allowing detection of a high quantity of proteins with the proteomic approach has been developed (Sarry et al., 2004). In this method, a solution containing 12.5% of trichloroacetic acid (TCA) in cold acetone and a reducing agent is used (Table 7.2). Using this approach only proteins in the pulp are extracted, and enzymes potentially involved in protein modification are inhibited. 

Thus, the differential expression of genes is required for the production of different proteins because each protein controls a distinct function. The function of many proteins is listed in Table 1.1. In addition, the protein profile of a cell can vary depending on the different kinds of modification of the same protein such modifications of protein may involve acetylation, phosphorylation, glycosylation, or association with lipid or carbohydrate molecules. These modifications in proteins occur as posttranslational events and alter the function of proteins. One example is the mitosis activator protein (MAP) kinase protein controlling the mitosis this protein is activated by phosphorylation to give MAP Kinase (MAPK), MAP kinase kinase (MAPKK), and MAP kinase kinase kinase (MAPKKK). The role of protein modification in the control of cellular activity is discussed later in this book. 

One class of mechanism-based MAO inhibitors includes the unsaturated alkylamines (propargylamine analogs) (Table II). Although the kinetics of enzyme inactivation for these compounds are consistent with a mechanism-based inhibitor, in only a few cases has the chemical mechanism and site of protein modification been determined. Pargyline (iV-benzyl-N-methyl-2-propynylamine) is a classic example. Pargyline reacts stoichiometrically and irreversibly with the MAO of bovine kidney, with protection from inactivation afforded by substrate benzylamine (91). Furthermore, the reaction involves bleaching of the FAD cofactor at 455 nm and the formation of a new absorbing species at 410 nm and a covalent adduct of inactivator with flavin cofactor (92). 

Of the many actual and potential uses of enzymes for chemical modification and improvement of proteins, that of hydrolysis of proteins is the most widely used. Hydrolysis involves the action of selected proteolytic enzymes to split specific peptide bonds in a protein. Along with a decrease in size of the protein there are changes in the solubility and functional properties of the product. Some of the uses of proteolytic enzymes in protein modification are shown in Table I. 

The concept of protein modification by PEGylation is now well established clinically (5,136,137), and is used to increase protein solubility and stability, reduce immunogenicity, prevent rapid renal clearance of small proteins, and prevent receptor-mediated clearance by cells of the reticuloendothelial system. Many HPMA copolymer-antibody, -protein, and-peptide conjugates have also been synthesized (Table 2). In the main, protein incorporation has been used to promote cell-specific drug targeting, but conjugates of biologically active proteins, enzymes and coiled-coil peptide domains have also been reported. 

PROTEINS AND PEPTIDES POSTTRANSLATIONAL MODIFICATIONS TABLE 9.1. Selected Posttranslational Modifications of Proteins... 

Biopharmaceuticals CE Analysis Table 2 Common protein modification and degradation pathways. 249... 

The structures and hydrolysis and aminolysis rates of some investigated M-PEG and other esters used in protein modification are shown in Table 1. Rates of reaction are influenced both by the nature of the leaving group and the pKa of the acid moiety. In the rates of biotin active esters, the koH- in min (i.e. rate constants for hydroxide caused hydrolyses) are 1.46 x lO for sulfo-N-hydroxysuccinimide, 2.28 x 10 for N hydroxysuccinimide, and 8.00 x 10 for hydroxy-2-nitrobenzene-4-sulfon-ic acid. The respective ti/2 s for hydrolysis are 26.9 min., 43.4 min., and 320 min. This order of magnitude difference is also reflected in the aminolysis rate constants (using as model amine 6-aminocaproic acid) which are 5.01 x 10, 7.71 x 10 and 3.16 x 10 min", respectively. The rate constant ratios for amine over hydroxide become 3.38, 3.43, and 3.95, which confirms that an improved ratio, and thereby better selectivity for protein amino groups can be obtained with an ester, such as HNSA, that reacts more slowly, and is less sensitive to buffer-catalyzed hydrolysis, as compared to N-hydroxysuccinimide ester. 

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