First isolated and characterized in 1900 by Gulewitsch, carnosine (beta-alanyl-L-hystidine) is a dipeptide commonly present in mammalian tissue, and in particular in skeletal muscle cells; it is responsible for a variety of activities related to the detoxification of the body from free radical species and the by-products of membrane lipids peroxidation, but recent studies have shown that this small molecule also has membrane-protecting activity, proton buffering capacity, formation of complexes with transition metals, and regulation of macrophage function. It has been proposed that carnosine could act as a natural scavenger of dangerous reactive aldehydes from the degradative oxidative pathway of endogenous molecules such as sugars, polyunsaturated fatty acids (PUFAs) and proteins. In particular, it has been recently demonstrated that carnosine is a potent and selective scavenger of alpha,beta-unsaturated aldehydes, typical by-products of membrane lipids peroxidation and considered second messengers of the oxidative stress, and inhibits aldehyde-induced protein-protein and DNA-protein cross-linking in neurodegenerative disorders such as Alzheimer's disease, in cardiovascular ischemic damage, in inflammatory diseases. The research for new and more potent scavengers for HNE and other alpha,beta-unsaturated aldehydes has produced a consistent variety of carnosine analogs, and the present review will resume, through the scientific literature and the international patents, the most recent developments in this field.

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: (a) the nature of the phosphorylatable amino acid, if any; (b) its position relative to the critically required acidic residues; (c) the extension and (d) the structure of the acidic cluster. All of them were tested as substrates and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined.The results suggest the following conclusions. (a) Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. (b) In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. (c) The affinity for the protein-binding site, as deduced from K , and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. (d) An acidic residue at position + 3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar K , but negligible V,,, compared to Ser-Glu-Ala-Glu-GluGlu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.Among the factors determining the specificity of protein kinases, the amino acid sequence around the target residue has been shown in many instances to play a crucial role. The structural features responsible for such site recognition can be studied with the aid of synthetic peptide substrates, reproducing with suitable modifications those segments of the target proteins in whch the phosphorylatable amino acids are located. This kind of approach not only illuminates the mechanism by which protein kinases can recognize and modify their own targets, but also can provide information about the structure of the catalytic site. Using synthetic peptide substrates it could be shown that several classes of protein kinases, including those dependent on CAMP, cGMP, Ca'+/ calmodulin and Ca2+/phospholipid, preferentially phosphorylate sets of diversely sequenced peptides sharing, however, the inclusion of critically required basic residues (e.g. [l -61). On the other hand these basic peptides are not affected at all by type-2 casein kinases (CK-2), termed also casein kinases-TS or G [7]. This is a class of ubiquitous and multisubstrate protein kinases, independent of cyclic nucleotides and Ca' +, In order to obtain more information about the structural factors determining the site recognition and the phosphorylation efficiency of CK-2, ten different additional hexapeptides were considered. These dif...

The local specificity determinants for a T-cell protein tyrosine phosphatase (TC-PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T-cell PTP, deprived of its C-terminal down-regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C-terminal downregulatory site of pp60"'" (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp6OC-"'" and Tyr412 of ~51'~'). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal-growth-factor receptor protein tyrosine kinases, the phosphorylated loop I11 of calmodulin, and phosphoangiotensin I1 undergo either very slow or undetectable dephosphorylation, even if tested up to 1 pM concentration.The replacement of either Ser-P or 0-methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src-(412 -418), src-(414 -41 8) and abl-(390 -397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N-terminal side of phosphotyrosine, with special reference to position -3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur.By replacing the truncated form of TC-PTP with the full length TC-PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in K , values, accompanied by a more or less pronounced decrease in V, , , values. These data support the concept that the C-terminal regulatory domain of TC-PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.Phosphotyrosine accounts for only 0.01 -0.05% of cellular phospho amino acids in normal tissues [l], and for 1 -3% of phospho amino acids in cells transformed by oncogenic retroviruses [2]. It is, nevertheless, firmly believed that phosphorylation of proteins at tyrosyl residues plays a crucial role in signal transduction and in the control of cell growth,

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for CAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-ArgS-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.