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L Engström

Publications and source records attributed to L Engström.

At least 37 records · Page 2Linked to original sources
PubMed1993

Protein kinase assay using tritiated peptide substrates and ferric adsorbent paper for phosphopeptide binding.

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.

Adsorption↗
PubMed1993

Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues.

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.

Amino Acid Sequence↗
PubMed1993

Phospholipid-dependent and EGTA-inhibited protein kinase from maize seedlings.

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.

Animals↗
PubMed1992

Phase behaviour and molecular species composition of oat root plasma membrane lipids. Influence of induced dehydration tolerance.

Tolerance to dehydration induced by repeated water-deficit stress is well correlated to changes in the lipid composition of oat root cell plasma membranes. The molecular species of the two dominant phospholipids phosphatidylcholine and phosphatidylethanolamine were determined. The four major species were 16:0/18:2, 16:0/18:3, 18:2/18:2 and 18:2/18:3. In contrast to the large changes in plasma membrane lipid composition in other respects, induced tolerance resulted in very weak alterations concerning the phospholipid molecular species pattern. Only minor alterations, appearing as a decrease in the 18:3-containing lipids, occurred. Total lipids of microsomes and isolated plasma membranes of root cells were analysed by X-ray crystallography at different degrees of hydration. The lipid phase behaviour at different degrees of hydration was further confirmed by polarization microscopy. In the presence of excess water all membrane lipids adopted a reversed micellar configuration. The plasma membrane lipids from root cells with induced dehydration tolerance formed upon dehydration two coexisting lamellar structures. The importance of the phase behaviour at different degrees of hydration for the membrane properties and the relation to membrane composition is discussed.

Edible Grain↗
PubMed1992

A potential pitfall in protein kinase assay: phosphocellulose paper as an unreliable adsorbent of produced phosphopeptides.

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.

Adsorption↗
PubMed1992

Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.

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.

Animals↗
PubMed1992

Peroperative endotoxin concentrations in portal and peripheral venous blood in patients undergoing right hemicolectomy for carcinoma.

OBJECTIVE: To find out the incidence of systemic and portal endotoxaemia in a homogeneous group of patients with colonic cancer undergoing a standard operation, and to compare them with a control group with benign disease. DESIGN: Open study. SETTING: Department of Surgery, Stockholm Söderhospital. SUBJECTS: 15 of 17 consecutive patients admitted for right hemicolectomy for cancer, and four control subjects (one with angiodysplasia, and three with ulcerative colitis) who were to undergo right hemicolectomy and proctocolectomy. INTERVENTION: After mobilisation of the colon, 10 ml samples of blood were taken simultaneously from a mesocolic vein, and from an antecubital vein, for assay of endotoxin. RESULTS: Raised concentrations of endotoxin were found in the portal blood of 4 of the 15 patients with cancer, and in two of the four with benign disease. One of the four with cancer also had a raised value in peripheral blood. There was no correlation between increased endotoxin concentration and tumour size, Dukes' stage, or development of infective complications. CONCLUSION: Pronounced concentrations of endotoxin are found only rarely in patients undergoing right hemicolectomy for cancer. The most likely explanation is translocation of endotoxin through mucosa damaged by an ulcerating tumour.

Adult↗
PubMed1992

Peptide fragments of myelin basic protein as substrates of protein kinase C.

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.

Amino Acid Sequence↗
PubMed1991

Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

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.

Amino Acid Sequence↗
PubMed1990

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

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 Km for C3 was 2.6 microM 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.

Amino Acids↗
PubMed1988

Treatment of appendiceal abscess.

Appendiceal abscess occurring in 193 patients during a 14-year period is reviewed. Treatment was nonsurgical in 98 patients, with a complication rate of 3%. In the 95 surgically treated patients the complication rate was 32%. One patient died. 'Interval' elective appendectomy was performed in 80 cases and the complication rate was 23%. Among the 32 patients without surgery or in whom the appendix was not removed at laparotomy, the recurrence rate was 3%. No malignancy was found at colonic radiography in 84 conservatively treated patients. Based on these findings, the following strategy is recommended for management of appendiceal abscess. 1) Initially conservative treatment, with surgical intervention if this fails. 2) 'Interval' elective appendectomy not routinely undertaken. 3) For patients older than 40, follow-up with colonic radiography, possibly colonoscopy and, if necessary, also exploratory laparotomy to exclude intraabdominal malignancy.

Abscess↗
PubMed1987

Effect of phosphorylation in vitro of human fibrinogen with protein kinase C on thrombin-induced gelation.

Thrombin-induced gel formation of fibrinogen phosphorylated by protein kinase C yielded a transparent gel, whereas unphosphorylated fibrinogen yielded a coarse gel. The mass-length ratio was found to be one order of magnitude higher for the unphosphorylated than for the phosphorylated fibrinogen. Since the phosphorylated sites are located near the cross-linking sites in the A alpha-chain of fibrinogen, it is likely that the introduction of charged phosphate groups in this region prevent the lateral growth of the fibrin fibres.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

The kinetic effects of in vitro phosphorylation of rabbit muscle enolase by protein kinase C. A possible new kind of enzyme regulation.

Rabbit muscle enolase was found to be phosphorylated in vitro by calcium-activated phospholipid-dependent protein kinase. The extent of incorporation (about 0.6 mol/mol subunit) and the apparent Km for the reaction (3.0 microM subunit) were determined. Kinetic studies on the unphosphorylated and phosphorylated enzymes displayed an unexpected effect of phosphorylation resulting in activation of the forward reaction and inhibition of the backward one.

Animals↗