| 1. |
- Järv, Jaak, et al.
(author)
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Quantitative structure-activity relationships in protein kinase C reaction with synthetic peptides derived from myelin basic protein
- 1996
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In: Bioorganic chemistry (Print). - : Elsevier BV. - 0045-2068. ; 24:2, s. 159-168
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Journal article (peer-reviewed)abstract
- A set of peptides, Lys-Arg-Pro-Ser-X-Arg-Ala-Lys-Ala, where X stands for Ala, Val, Leu, Ile, Phe, Pro, Lys, Arg, Asp, Glu, Asn, Gln, and His, was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. All compounds, except the peptide with Pro at the position X, were effectively phosphorylated by this enzyme, and for these substrates the kinetic constantsKm, maximal velocity constantsV, and second-order rate constantskIIwere determined. The data were analyzed by means of quantitative structure–activity relationships, taking into account hydrophobicity of the variable amino acids, bulkiness of their side groups quantified by molecular refractivity constants MR, and ionic status of these substituents by using an independent variable +1 for cationic, −1 for anionic, and 0 for nonionic substituents. These structural factors influenced theKmvalues, while the maximal velocity of phosphorylation depended mostly on the ionic status of the variable amino acid. The latter effect seems to characterize electrostatic interaction between the substrate molecule and some negative charge located in the enzyme active center.
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| 2. |
- Bergström, Gunnel, et al.
(author)
-
Proteolytic modification of pig and rat liver pyruvate kinase including the phosphorylatable site
- 1978
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In: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 532:2, s. 259-267
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Journal article (peer-reviewed)abstract
- The phosphorylated or phosphate-accepting site of pyruvate kinase from pig and rat liver was removed without inactivation by incubation with subtilisin. At different time intervals the subtilisin was inactivated with phenylmethylsulfonyl fluoride and the amount of remaining phosphorylatable or phosphorylated sites of pyruvate kinase estimated by incubation with an excess of [32P]-ATP and protein kinase. It was found that to get the same rate of modification the subtilisin concentration required to modify unphosphorylated pyruvate kinase was approximately ten times higher than that used for removal of the phosphorylated site of phosphorylated site of phosphorylated enzyme. It was shown that the proteolytically-modified pyruvate kinase had an increased apparent Km for phosphoenolpyruvate without a change in V, when compared to unmodified unphosphorylated and phosphorylated pyruvate kinase. The removal of the phosphorylated site was not associated with loss of the allosteric sites for ATP and Fru-1,6-P2. The possibility that phosphorylation of the pyruvate kinase increases its degradation rate in vivo is briefly discussed.
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| 3. |
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| 4. |
- Dahlqvist, Ulla, et al.
(author)
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Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions
- 1978
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 540:1, s. 13-23
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Journal article (peer-reviewed)abstract
- Liver cell sap from normally fed rats, rats fed with a high-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42000, 21000, 52000 and 49000. The component with a minimal molecular weight of 42000 seemed to have a native molecular weight of 160000. Both the 21000 and the 52000 component had a native molecular weight of about 110000-120000. The protein with a minimal molecular weight of 49000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54000, 39000, 34000 and 22000.
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| 5. |
- Edlund, Bror, et al.
(author)
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Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase
- 1975
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In: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 67:4, s. 1516-1521
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Journal article (peer-reviewed)abstract
- One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.
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| 6. |
- Ek, Pia, et al.
(author)
-
Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3´, 5´-AMP-stimulated protein kinase
- 1976
-
In: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 429:2, s. 374-382
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Journal article (peer-reviewed)abstract
- The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.
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| 7. |
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| 8. |
- Ekman, Pia, et al.
(author)
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The quantity of protein-bound (32P)phosphotyrosine in hepatocytes and fibroblasts : The effects of tyrosine protein kinase activating agents
- 1987
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In: Journal of Biochemistry (Tokyo). - 0021-924X .- 1756-2651. ; 101:4, s. 863-870
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Journal article (peer-reviewed)abstract
- Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.
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| 9. |
- Ekman, Pia, et al.
(author)
-
Two methods to avoid the effect of endogenous inhibitors during the assay of protein kinase C activity in tissue extracts.
- 1992
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In: Preparative Biochemistry. - : Informa UK Limited. - 0032-7484. ; 22:2, s. 165-175
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Journal article (peer-reviewed)abstract
- Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.
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| 10. |
- Eller, Marika, et al.
(author)
-
Peptide fragments of myelin basic protein as substrates of protein kinase C.
- 1992
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In: Biochemistry International. - 0158-5231. ; 27:4, s. 625-631
-
Journal article (peer-reviewed)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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| 11. |
- Eller, Marika, et al.
(author)
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Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides
- 1991
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In: Biochemistry International. - 0158-5231. ; 25:3, s. 453-460
-
Journal article (peer-reviewed)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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| 12. |
- Eller, Marika, et al.
(author)
-
Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues.
- 1993
-
In: Journal of Biochemistry. - 0021-924X. ; 114:2, s. 177-180
-
Journal article (peer-reviewed)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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| 13. |
- Engström, Lorentz, et al.
(author)
-
Pyruvate kinase
- 1987
-
In: The enzymes. - : Elsevier. - 9780121227180 ; , s. 47-75
-
Book chapter (peer-reviewed)
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| 14. |
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| 15. |
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| 16. |
- Forsberg, Per-Olof, et al.
(author)
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In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C : Effects on the classical and alternative pathways
- 1990
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:5, s. 2941-2946
-
Journal article (peer-reviewed)abstract
- Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Kmfor C3 was 2.6 µM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.
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| 17. |
- Humble, Elisabet, et al.
(author)
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Amino acid sequence at the phosphorylated site of rat liver fructose-1,6-diphosphatase and phosphorylation of a corresponding synthetic peptide
- 1979
-
In: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 90:3, s. 1064-1072
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Journal article (peer-reviewed)abstract
- Rat liver fructose-1,6-diphosphatase was phosphorylated with (32P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(32P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent Km of 400 μM, while the apparent Km value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The Vmax value was 20 times higher for the peptide than for the enzyme.
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| 18. |
- Järv, Jaak, et al.
(author)
-
Phosphorylation of Sepharose-coupled peptides by protein kinase A
- 1996
-
In: Bioorganic chemistry. - : Elsevier BV. - 0045-2068. ; 24:1, s. 1-9
-
Journal article (peer-reviewed)abstract
- The kinetics of phosphorylation of the Sepharose-coupledpeptide RRASVA by catalytic subunit of proteinkinaseA, and diastereomers of this peptide, containingd-amino acids successively in each position, were studied. Coupling of these peptides with the amino and carboxyl termini to CH- and AH-Sepharoses had similar effects on the phosphorylation reaction, increasing theKmand decreasing theVvalues, respectively. The diastereomeric peptides were also phosphorylated by the enzyme and the rate of this reaction depended on the position of substitution ofl-amino acids with theird-analogs. However, this dependence was much less pronounced if compared with stereoselectivity of the enzyme in reactions with these peptides in solution: theKmvalues for the Sepharose-coupledpeptides were almost insensitive to the replacement ofl-amino acids withd-analogs and moderate stereoselectivity was revealed in the maximal velocity of the reaction. The Sepharose-coupledpeptide containingd-serine was also phosphorylated by proteinkinaseA while the same peptide in solution did not interact with the enzyme. Consequently, the polymer, enveloping the phosphorylatable peptide, may remarkably influence the recognition of the reaction site, altering both V and Km values.
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| 19. |
- Loog, Mart, et al.
(author)
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Comparision of substrate specificities of protein kinases A and C based on peptide substrates
- 1994
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In: Bioorganic chemistry. - : Elsevier BV. - 0045-2068. ; 22:3, s. 328-336
-
Journal article (peer-reviewed)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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| 20. |
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| 21. |
- Muszynska, Grazyna, et al.
(author)
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Phospholipid- dependent and EGTA-inhibited protein kinase from maize seedlings
- 1993
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In: Biochemistry and Molecular Biology International. - 1039-9712. ; 30:5, s. 849-860
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Journal article (peer-reviewed)abstract
- Chromatography of a maize seedling extract on DEAE-cellulose, followed by Octyl-Sepharose yielded a fraction with protein kinase activity which was stimulated by phosphatidylserine plus diolein. The activity was not enhanced by calcium ions but was inhibited by chelating agents and could then be restored by the addition of calcium ions. All these facts indicated that the maize protein kinase was similar to mammalian protein kinase C. The maize enzyme phosphorylated myelin basic protein (MBP) and histone H1, but the MBP-peptide4-14 and protamine were poor substrates for the enzyme. Further purification of the enzyme fraction followed by phosphorylation and SDS-polyacrylamide gel electrophoresis, revealed two labeled bands of Mw 59 and 83 kDa the former of which probably being the protein kinase C.
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| 22. |
- Toomik, Reet, et al.
(author)
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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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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 204:2, s. 311-314
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Journal article (peer-reviewed)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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| 23. |
- Toomik, Reet, et al.
(author)
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D-amino acid residues as substrate specificity determinants for casein kinase II
- 1994
-
In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 200:3, s. 1564-1569
-
Journal article (peer-reviewed)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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| 24. |
- Toomik, Reet, et al.
(author)
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Protein kinase assay using tritiated peptide substrates and ferric adsorbent paper for phosphopeptide binding
- 1993
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 209:2, s. 348-353
-
Journal article (peer-reviewed)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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| 25. |
- Trojanek, Joanna, et al.
(author)
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Phosphorylation of plant proteins and the identification of protein - tyrosine kinase activity in maize seedlings
- 1996
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 235:1-2, s. 338-344
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Journal article (peer-reviewed)abstract
- Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).
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