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F Marchiori

Publications and source records attributed to F Marchiori.

At least 37 records · Page 2Linked to original sources
PubMed1986

The substrate specificity of the protein kinase induced in cells infected with herpesviruses: studies with synthetic substrates [corrected] 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.

Animals↗
PubMed1986

Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates.

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: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. 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. The affinity for the protein-binding site, as deduced from Km 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. 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 Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu 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.

Amino Acids↗
PubMed1985

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 mR 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 mR 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 km values with TK-I and TK-II, (100 and 10 microM, respectively). When TK-I was incubated with [gamma-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.

Animals↗
PubMed1985

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 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-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.

Animals↗
PubMed1985

Synthetic and binding studies on the calcium binding site I of bovine brain calmodulin. II. Synthesis and CD studies on the cyclic 20-31 sequence.

The synthesis of the cyclic 20-31 sequence of bovine brain calmodulin corresponding to the loop of the hypothetical calcium binding site I of the protein has been accomplished by classical solution methods. The interaction of the synthetic cyclic fragment with calcium ions has been investigated by CD spectroscopy in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of aqueous solution of the cyclic dodecapeptide in the wavelength range 200-250 nm. In 98% trifluoroethanol the CD spectrum of the cyclic compound in the absence of calcium ions is almost identical to that of the linear dodecapeptide in the presence of saturation concentrations of calcium. This result supports our previous hypothesis of a folding of the linear sequence upon interaction with the metal ion. The cyclic peptide also interacts with calcium ions in 98% trifluoroethanol forming a 1:1 complex.

Amino Acid Sequence↗
PubMed1984

Synthetic peptides including acidic clusters as substrates and inhibitors of rat liver casein kinase TS (type-2).

The hexapeptides AcSer-Glu-Glu-Glu-Val-Glu and Ser-Glu-Glu-Glu-Glu-Glu, reminiscent of the sites phosphorylated by type-2 casein kinase TS in troponin T and glycogen synthase, respectively, have been synthesized and tested as phosphorylatable substrates for casein kinase TS as well as for other protein kinases. Both peptides are readily phosphorylated by casein kinase TS but not, to any detectable extent, by either cAMP-dependent protein kinase or phosphorylase kinase. Phosphorylation by type-1 casein kinase S was almost negligible. On the other hand the hexapeptide Ser-Glu-Glu-Glu-Ala-Ala is phosphorylated much more slowly and the hexapeptide Ser-Glu-Glu-Ala-Ala-Ala is almost unaffected by casein kinase TS. While the Vmax values of casein kinase TS with the acidic hexapeptides are comparable to those obtained with the corresponding protein substrates, the apparent Km values for the peptides are about two orders of magnitude higher than those for the protein substrates. The heptapeptide Arg-Ser-Glu-Glu-Glu-Val-Glu is a very poor substrate of casein kinase TS in comparison with the corresponding hexapeptide lacking the N-terminal Arg; it is, however, a competitive inhibitor toward the protein substrates, exhibiting a Ki similar to those of Ser-Glu-Glu-Glu-Glu-Glu and (Glu)5 which, in turn, are one order of magnitude higher than that of (Glu)10. It is concluded that the minimum structural requirement of type-2 casein kinases consists of a phosphorylatable residue followed by an acidic cluster, whose length is critical for the binding to the enzyme. Additional residues on the N-terminal side are not required, but their nature can influence the transphosphorylation reaction considerably.

Amino Acid Sequence↗
PubMed1984

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.

Animals↗
PubMed1984

Crystal structure of cyclo-L-histidyl-L-methionine and studies of metal complexation with divalent metal ions.

The structure of the title compound displays a buckled diketopiperazine ring. The two side-chains are both folded, with the imidazole ring facing the methionine chain. Imidazole nitrogen is the primary site for metal coordination of the ligand. Only for copper (II) adducts and in the solid state are there evidences of additional coordination through the sulfur atom.

Cations, Divalent↗
PubMed1983

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 (Ki 0.11 microM). (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.

Adenosine Triphosphate↗
PubMed1983

Synthetic peptides reproducing the site phosphorylated by cAMP-dependent protein kinase in protein phosphatase inhibitor-1. Effect of structural modifications on the phosphorylation efficiency.

The hexapeptide (Arg)2-Pro-Thr-Pro-Ala (T1) and octapeptide (Arg)2-Pro-Thr-Pro-Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor-1, a physiological target of cAMP-dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP-dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2-Ala-Ser-Val-Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C-terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N-terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T1 on its C-terminal side to give the derivative (Arg)2-Pro-Thr-Pro-Ala-Thr-Val-Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor-1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.

Amino Acids↗
PubMed1983

Synthetic and binding studies on the postulated calcium binding site I of calmodulin.

The synthesis of the dodecapeptide [sequence (20--31)] representing the hypothetical calcium binding site I of calmodulin by classical methods in solution is described. The interaction of this synthetic calmodulin fragment with calcium ions has been investigated by CD in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of the dodecapeptide in aqueous solution in the wavelength range 200-250 nm. However, in 98% trifluoroethanol a linear variation of the CD properties has been observed as a function of the Ca2/peptide molar ratio. The CD results indicate the formation of a 1 : 1 calcium-dodecapeptide complex with a very high binding constant.

Amino Acid Sequence↗
PubMed1982

Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase.

The synthetic phosphohexapeptides Arg-Arg-Ala-Thr(35P)-Val-Ala and Arg-Arg-Ala-Ser(32P)-Val-Ala, phosphorylated by the cAMP-dependent protein kinase and differing only in the nature of the phosphorylated residue, have been used as substrates of a partially purified rat liver protein phosphatase-T, distinct from the multifunctional protein phosphatase-1. While the phosphothreonyl hexapeptide is readily dephosphorylated (exhibiting a Km = 15 microM), the phosphoseryl one is almost unaffected. Such a behavior is not shared by protein phosphatase-1, calf intestine alkaline phosphatase, and potato acid phosphatase, all of which are more active on the phosphoseryl hexapeptide. The NH2-terminal basic residues critical for cAMP-dependent phosphorylation are not required in the dephosphorylation reaction, as both Arg can be removed without impairing the efficiency of protein phosphatase-T toward the phosphothreonyl peptide. On the other hand, the replacement of 2 Pro for the Ala and Val flanking Thr(32P), to give a new phosphohexapeptide reproducing the phosphorylated site of protein phosphatase inhibitor-1, prevents the protein phosphatase-T activity. Moreover, IgG heavy chain 32P labeled in tyrosine is not affected by protein phosphatase-T, while it is dephosphorylated by alkaline phosphatase. These results would indicate that protein phosphatase(s)-T represent a distinct class of protein phosphatases specifically involved in the dephosphorylation of phosphothreonyl residues fulfilling definite structural requirements.

Acid Phosphatase↗
PubMed1982

Coordination chemistry of peptides. Part II. Crystal structure of cyclo-L-methionylglycine and studies of metal complexation.

The structure of the title compound displays a nearly planar diketopiperazine ring. The side chain is folded on the ring, the S atom making a significantly short contact with the methionine carbonyl atom. The crystal packing shows an unusual assembly of alternating layers of peptide and crystallization water molecules. The side chain sulfur atom is found to be the primary site for metal coordination of the ligand, while in some systems there is evidence of additional coordination through deprotonated amide nitrogens.

Dipeptides↗
PubMed1981

Synthesis of leupeptins and inhibition of proteinases. I. Inhibition of acrosin and trypsin.

A series of leupeptin analogs R-L-leucyl-L-leucyl-L-argininal with variable N-terminal substituents has been synthesized using N alpha-tert-butyl-oxycarbonyl-NG-benzyloxycarbonyl-L-arginine-delta-lactam as the starting material. The modified leupeptins proved to be strong competitive inhibitors of the endoprotease acrosin from mammalian spermatozoa. Inhibition constants were found in the range of 4.7 X 10(-7)M (R = H) to 9.7 X 10(-9)M (R = tert-butyloxycarbonyl). N alpha-tert-butyloxycarbonyl leupeptin represents the strongest acrosin inhibitor synthesized so far. Two of the leupeptin derivatives (R = trifluoroacetyl, R = tert-butyloxycarbonyl) were more effective than the natural leupeptins from microbial sources (Ki = 5.9 X 10(-8)M). The potential use of synthetic leupeptins as antienzymatic contraceptives is discussed.

Acrosin↗