| 1. |
- Eller, Marika, et al.
(författare)
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Peptide fragments of myelin basic protein as substrates of protein kinase C.
- 1992
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Ingår i: Biochemistry International. - 0158-5231. ; 27:4, s. 625-631
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Tidskriftsartikel (refereegranskat)abstract
- A set of peptides derived from myelin basic protein was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. The replacement or the removal of the N-terminal Gln had no effect on the activity of the parent peptide. The removal of the following Lys or Arg led to a systematic decrease in substrate activity. The modifications in the C-terminal part of the peptide had a weaker influence on the parameters Vmax and KM than those in the N-terminal. The rather regular dependence of the activity of substrates upon their structure does not allow the strict definition of a minimum substrate for protein kinase C.
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| 2. |
- Eller, Marika, et al.
(författare)
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Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides
- 1991
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Ingår i: Biochemistry International. - 0158-5231. ; 25:3, s. 453-460
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Tidskriftsartikel (refereegranskat)abstract
- A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.
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| 3. |
- Eller, Marika, et al.
(författare)
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Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues.
- 1993
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Ingår i: Journal of Biochemistry. - 0021-924X. ; 114:2, s. 177-180
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Tidskriftsartikel (refereegranskat)abstract
- A set of stereoisomeric nonapeptides KRPSQRAKY with one, two, or all L-amino acid residues replaced by the corresponding D-amino acids, and two analogs with L- and D-threonine instead of serine, were synthesized and tested as substrates for protein kinase C. All of the peptides were phosphorylated by the enzyme. The maximal rate of the reaction with the all-D peptide was more than one order of magnitude lower than that for all-L peptide with serine. The same applied to the peptides with D-Ser or with D-Arg in position +2 with respect to Ser. The Km values for the peptides containing one D-amino acid were close to that for the prototype peptide (53 microM). On the other hand, when two or more D-amino acids were present, the Km value increased considerably. Replacement of serine by threonine also reduced the phosphorylation rate and increased the Km values. One can conclude that the stereospecificity of protein kinase C is much less pronounced than that of protein kinase A, which is in agreement with the less clearly pronounced substrate specificity of the former enzyme.
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| 4. |
- Loog, Mart, et al.
(författare)
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Comparision of substrate specificities of protein kinases A and C based on peptide substrates
- 1994
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Ingår i: Bioorganic chemistry. - : Elsevier BV. - 0045-2068. ; 22:3, s. 328-336
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Tidskriftsartikel (refereegranskat)abstract
- Peptides, obtained by gradual removal of amino acids from both ends of pEKRPSQRSKYL, and stereoisomeric nonapeptides KRPSQRAKY with one D-amino acid residue successively in each position, were tested as substrates for protein kinase A, All these compounds were phosphorylated but at quite different rates by the enzyme. Comparison of the kinetic data with the appropriate results for protein kinase C, measured earlier, was used to analyze and compare the specificity determining factors of these enzymes. The analysis of the cross-specificity points to the possibility that only a short part, mainly the sequence of 1 to 2 amino acids around the phosphorylatable serine residue, is important for differentiation of substrates by these enzymes, while the remaining part of the peptide structure has similar influence on their reactivity in the case of these two protein kinases. Thus, the active center of these enzymes can be conventionally divided into two parts, which are responsible for selectivity and effectiveness of the phosphorylation reaction, respectively.
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| 5. |
- Toomik, Reet, et al.
(författare)
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A potential pitfall in protein kinase assay: phosphocellulose paper as un unreliable adsorbent of produced phosphopeptides
- 1992
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 204:2, s. 311-314
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Tidskriftsartikel (refereegranskat)abstract
- The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.
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| 6. |
- Toomik, Reet, et al.
(författare)
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D-amino acid residues as substrate specificity determinants for casein kinase II
- 1994
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 200:3, s. 1564-1569
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Tidskriftsartikel (refereegranskat)abstract
- A set of diastereomeric peptides RRRDDDSDDD each with a corresponding D-amino acid residue successively in every position (except the arginines) was tested as substrates for casein kinase II. It was found that the D-serine containing peptide was not detectably phosphorylated. The replacements at the positions +1, -1 and +2 decreased the kII values more than 60, 30 and 20 times, respectively. The effect of the L/D replacements decreased with increased distance from the serine. The D-amino acid scan used herein seems to be a helpful complementary tool for studies of substrate specificity determinants for different protein kinases.
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| 7. |
- Toomik, Reet, et al.
(författare)
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Protein kinase assay using tritiated peptide substrates and ferric adsorbent paper for phosphopeptide binding
- 1993
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 209:2, s. 348-353
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Tidskriftsartikel (refereegranskat)abstract
- The recently described synthesis of ferric adsorbent paper has made possible the modification of protein kinase assays. The adsorbent contains ferric chelate groups, which are responsible for the binding of phosphopeptide via phosphate group. The selective adsorption of phosphopeptide contra nonphosphorylated peptide allows the use of tritium-labeled peptides and unlabeled ATP as substrates. The binding of the reaction product to the adsorbent was complete and was not affected by the amino acid sequence of the peptide. The conditions required for the separation of the produced phosphopeptide from the initial peptide have been worked out as well. The firmly bound phosphopeptide should be released from the ferric adsorbent paper prior to liquid scintillation counting. Using 0.1 m NH4HCO3 solution (pH 8.0), the elution of phosphopeptides was almost complete. The modified protein kinase assay proposed herein is rapid and allows handling of multiple samples simultaneously. In addition, the ferric paper method avoids the use of 32P-isotope, replacing it with 3H which has lower radiation energy and a much longer half-life.
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| 8. |
- Toomik, Reet
(författare)
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Substrate specificity of protein kinases studied with synthetic peptides
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concentrates on the substrate specificities of protein kinases A and C (PKA, PKC). Synthetic peptides are used for that purpose, both in soluble and cellulose membrane-bound form. The, stereospecificity of both protein kinases was studied with D-amino acid containing synthetic peptides.The reactions of protein kinases with peptides are often monitored by adsorbing the phosphorylatedpeptides onto phosphocellulose paper, an anionic adsorbent. The presence of at least two basicamino acid residues in the peptide has been considered to be sufficient for the adsorption. In thisthesis, it was found that none of the phosphopeptides studied were adsorbed completely and therecovery of the phosphopeptide could vary twofold even if the peptides contained an equal numberof basic residues.A PKC substrate peptide pEKRPSQRSKYL was gradually shortened from either end. Themodifications at the C-terminus had a weaker influence on the reaction parameters than those at theN-terminus. The less pronounced specificity of PKC compared to that of PKA did not permit us todefine a minimum length substrate for this enzyme.The stereospecifically sensitive regions he in different parts of the substrates for PKA (RRASVA)and PKC (KRPSQRAKY). In the case of PKA, major effects appeared at the N-terminal side of thephosphorylatable serine while the most sensitive part in PKC substrates lies at the C-terminal side.PKA has a strong requirement for L-configuration at the serine residue while PKC can recognizeboth D- and L-configurations at the phosphorylation center. PKA has a well-defined core regionsensitive to changes in the substrate (-RAS-). In the case of PKC the stereochemical requirementsaround the phosphorylatable residue are not as strong. In conclusion, the stereospecificity of PKC isnot as clearly pronounced as that of PKA.In this thesis, it has been shown that SPOTs-membrane-bound peptides can be used for evaluation ofprotein kinase substrates. Using that method combined with the analysis of soluble peptides wefound a substrate peptide for PKC which was both sensitive (Km = 0.5 μM) and selective, as itshowed low or negligible cross-reactivity with other kinases tested.
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