Fernando Marchiori

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.

The substrate specificity of the protein kinase induced in cells infected with herpesviruses: Studies with synthetic substatres indicate structural requirements distinct from other protein kinases

Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with alpha-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simplex virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different.

AN INVESTIGATION OF THE SUBSTRATE SPECIFICITY OF PROTEIN PHOSPHATASE 2C USING SYNTHETIC PEPTIDE SUBSTRATES ; COMPARISON WITH PROTEIN PHOSPHATASE 2A

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.

Isolation and partial characterization of distinct forms of tyrosine protein kinases from rat spleen

Three peaks of tyrosine protein kinase activity (TK‐I, TK‐II and TK‐III) can be resolved when the extract of rat spleen particulate fraction is subjected to DEAE‐cellulose gradient chromatography. TK‐I and TK‐II, insensitive to both EGF and insulin, have been further purified by Sephacryl S200 gel filtration and characterized. TK‐I has an apparent m r of 65000, by far prefers Mn2+ over Mg2+ as activator, can use GTP besides ATP as phosphate donor and is stimulated 2‐3‐fold by polylysine. TK‐II, whose m r approximates 50000, is equally activated by Mg2+ and Mn2+, does not use GTP and is insensitive to polylysine. TK‐I and TK‐II can phosphorylate the synthetic peptide Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly (as well as its derivative with Orn in place of Arg), angiotensin II and poly(Glu, Tyr) 4:1 which exhibits different k m, values with TK‐I and TK‐II, (100 and 10 μM, respectively). When TK‐I was incubated with [γ‐32P]ATP and MnCl2 a doublet of alkali‐stable radiolabeled bands with molecular masses of 55 and 60 kDa were observed. Under identical conditions TK‐II gives rise to a single alkali‐stable radiolabeled band of 51 kDa, which may represent the autophosphorylation product of TK‐II itself.

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.

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-¿Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]¿ [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.

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.

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