| 1. |
- Ekman, Pia
(författare)
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Dephosphorylation with alkaline phosphatase of histone and fibrinogen phosphorylated with protein kinase C in vitro.
- 1991
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Ingår i: Upsala Journal of Medical Sciences. - : Scandinavian university press. - 0300-9734 .- 2000-1967. ; 96:2, s. 95-102
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline phosphatase from calf intestinal mucosa dephosphorylated histone H1 and fibrinogen that had been phosphorylated with protein kinase C. The reaction velocity was dependent on the ionic strength of the buffer; decreasing with increasing concentration. The pH optimum was around 7, which is lower than pH-optima described for other kinds of substrates. (32P) phosphorylated fibrinogen was dephosphorylated about 20 times faster than (32P)phosphohistone on a weight basis and the reaction continued linearily with time for the longest time tested (3 hs) even at 37 degrees C. As alkaline phosphatase is present in the blood the possible physiological significance of the dephosphorylation of phosphofibrinogen is discussed.
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| 2. |
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| 3. |
- Ekman, Pia, et al.
(författare)
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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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Ingår i: Preparative Biochemistry. - : Informa UK Limited. - 0032-7484. ; 22:2, s. 165-175
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Tidskriftsartikel (refereegranskat)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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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Eriksson, Stefan, et al.
(författare)
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Endothelial cells release casein kinase II like activity capable of phosphorylating fibrinogen in response to thrombin
- 1993
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 72:4, s. 315-320
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Tidskriftsartikel (refereegranskat)abstract
- Rat liver endothelial cells cultivated in the absence of serum and activated with thrombin released up to 10% of the total protein kinase activity into the cell medium using casein or fibrinogen as the phosphate acceptor protein. The activity was partly inhibited by heparin, indicating that it was of the casein kinase II type. The release of kinase started directly after the addition of thrombin (2 NIH U/ml) to the media with two maxima; one after about 10 min and the second after around 30 min. The phosphorylating activity of media from cells incubated for longer times was less dependent on thrombin-induction which probably indicated the start of destruction of the cells. The results reported suggest that phosphorylation of fibrinogen could occur in the blood under acute phase conditions.
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| 8. |
- Forsberg, Per-Olof, et al.
(författare)
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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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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:5, s. 2941-2946
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Tidskriftsartikel (refereegranskat)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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| 9. |
- 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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| 10. |
- Martin, Steven, et al.
(författare)
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Increased phosphate content of fibrinogen in vivo correlates with alteration in fibrinogen behaviour
- 1992
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 68:6, s. 467-473
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Tidskriftsartikel (refereegranskat)abstract
- Fibrinogen was purified from five patients admitted for hip-replacement surgery the day before (day 0), the day after (day 2) and and one week after the operation (day 8). The behaviour of each patient's three fibrinogens was compared in thrombin gelation assays and plasmin degradation experiments to investigate whether the reported increase in protein-bound phosphate at day 2 and day 8 had any effect on the functional behaviour of fibrinogen as has been demonstrated in vitro. It was found that the thickness of the fibrin fibres produced by thrombin increased markedly at day 2 and declined thereafter. Susceptibility to plasmin appeared to decrease post-operatively by 50% and remained at that level on day 8 despite the phosphate content returning to normal. This has also been shown for fibrinogen phosphorylated in vitro. We conclude, after testing the fibrinogens with and without alkaline phosphatase pretreatment, that our data most resemble the published findings for in vitro phosphorylation of fibrinogen by casein kinase II.
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