Gianfranco Borin

Carnosine and carnosine-related antioxidants: a review.

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 Alzheimers 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.

Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates.

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 tyrosyi 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‐Arg6‐Tyr, which is not a substrate for A‐kinase. Moreover, although the peptide Pro‐Arg5‐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.

Synthetic fragments of protamines as model substrates for rat liver cyclic AMP-dependent protein kinase☆

Nine synthetic peptides reproducing either exactly or with suitable substitutions three phosphorylatable sites of the protamines thynnine Z1 and galline have been prepared and tested as phosphate acceptors for the rat liver cyclic AMP-dependent protein kinase (type I). The most significant results obtained can be summarized as follows: 1. The hexapeptide: Arg-Arg-Ser-Thr-Val-Ala gives two phpsphorylated products, containing only Ser-P and both Ser-P and Thr-P, respectively. The relative amount of the di-phosphorylated derivative is not dependent on the incubation time but rather on the concentration of the substrates. 2. Both the replacements of ornithine for the two N-terminal arginines of glutamic acid for Val5 in the above peptide completely prevent phosphorylation, without conferring any inhibitory activity to the modified peptides, thus supporting the view that the N-terminal guanido groups and the C-terminal hydrophobic residue(s) are both required for the binding at the catalytic site rather than for the subsequent transphosphorylation reaction. 3. The replacement of alanine for Ser3 gives rise to a peptide whose Thr4 residue is still phosphorylated with an efficiency comparable to that of the unmodified peptide. The di-substituted peptide: Arg-Arg-Ala-Ser-Val-Ala however exhibits a dramatically lower Km value indicating that serine is a much better target than threonine whenever it is not adjacent to the N-terminal arginine couple. 4. The importance of the distance between the target residue and the N-terminal basic determinants is also evidenced by the phosphorylation of the dodecapeptide: Pro-(Arg)5-Ser-Ser-Arg-Pro-Val-Arg exhibiting a Km value about 20-times higher than that of salmine A1, whose phosphorylation site is comprised within an identical amino acid sequence, including however three rather than two adjacent serine residues. 5. The tetradecapeptide: (Arg)4-Tyr-Gly-Ser-(Arg)6-Tyr is completely unaffected by the kinase though a very similar site is found phosphorylated in native iridines, probably through a cyclic AMP-independent mechanism.

Opposite and mutually incompatible structural requirements of type-2 casein kinase and cAMP-dependent protein kinase as visualized with synthetic peptide substrates.

The synthetic hexapeptide Ser‐Glu‐Glu‐Glu‐Val‐Glu and its N‐acetylated derivative are readily and specifically phosphorylated by rat liver casein kinase TS (type‐2), while the derived heptapeptide with an additional N‐terminal Arg is a very poor substrate. Conversely, the substitution of Glu for Val5 in the synthetic peptide Arg‐Arg‐Ser‐Thr‐Val‐Ala, which is a good substrate for cAMP‐dependent protein kinase by virtue of the N‐terminal arginyl residues, prevents its phosphorylation by this enzyme. These data indicate that the site specificities of these two classes of protein kinases, requiring acidic and basic residues on the C‐and N‐terminal sides of the target residue(s), respectively, are mutually incompatible.

Polyglutamyl peptides: a new class of inhibitors of type-2 casein kinases

Casein kinase‐TS (Ck‐TS), a type‐2 casein kinase purified from rat liver cytosol which phosphorylates seryl and threonyl residues N‐terminal to acidic clusters, is specifically inhibited by polyglutamyl peptides which are ineffective both on type‐1 casein kinase and on cAMP‐dependent protein kinase. The inhibition is competitive toward the protein substrate and non‐competitive toward ATP. Among the polyglutamates tested (Glu)70 is the most effective (K i 0.11 μM). (Glu)10 and (Glu)5 are also inhibitors, though less powerful than (Glu)70, while (Glu)3, (Glu)2 and free glutamic acid up to 5 mM are ineffective. These results disclose the possibility that naturally occurring polypeptides containing long stretches of acidic residues may act as physiological inhibitors of type‐2 casein kinases.

Protamines: II. Circular dichroism study of the three main components of clupeine☆

The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.

Studies on cytochrome c. Part II. Synthesis of the protected heptapeptide (sequence 17–23) of Baker's yeast iso‐1‐cytochrome c

The synthesis is described of the N‐benzyloxycarbonyldecapeptide tert‐butoxycarbonylhydrazide, which corresponds to the sequence 57–66 of bakers yeast iso‐1‐cytochrome c. The peptide derivative was synthesized coupling two smaller subunits via the Rudinger modified azide procedure.

Relation between structure and function in some partially synthetic ribonucleases S′. I. Kinetic determinations

Abstract For a better understanding of the structure—function relationships in the ribonuclease molecule, detailed kinetic studies have been carried out on partially synthetic ribonucleases in which some amino acid residues were substituted or deleted. The following ribonuclease S′ analogs have been examined: [Orn10]-ribonuclease S′; des-Lys1-[Orn10]-ribonuclease S′; des-Lys1, Glu2-[Orn10]-ribonuclease S′; des-Lys1, Glu2, Thr3-[Orn10]-ribonuclease S′ and [Pro6, Orn10]-ribonuclease S′. In order to regenerate the arginyl residue, which is present in position 10 in the natural sequence, the S-peptide analogs belonging to the [Orn10]-series were transformed into the corresponding guanidinated derivatives by treatment with O-methylisourea. The activation curves of the S-protein with varying amounts of the S-peptide analog, before and after guanidination, against different substrates have been determined. Moreover, the kinetic parameters of the modified enzymes obtained by mixing S-protein with different S-peptide analogs were calculated. The hypothesis of the existance of an interaction between the γ-carboxyl group of glutamic acid in position 2 in the S-peptide sequence and the guanidinium group of arginine in position 10 has found experimental support. The absence of this interaction, which probably stabilizes the conformation of the N-terminal sequence of the enzyme, brings about some conformational changes on the active center region.

Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.

The influence of amino acid side-chains on α-helix stability: S-peptide analogues and related ribonucleases S′

Abstract In order to determine the influence of amino acid side-chains on α-helix stability, in relation to the protein folding process, the coil-helix transitions of some synthetic [Orn 10]-S-peptide analogues, containing, in position 8, Phe, Tyr, Ile, Ala, cpGly † and Gly, were investigated by the technique of circular dichroism under two different sets of conditions. First, the transitions of the Speptide analogues in water/trifluoroethanol mixtures were recorded. From the pattern of the transitions and from the ellipticity values in 97% trifluoroethanol, the following increasing order of amino acids as α-helix formers was found: Gly