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R Einarsson

Publications and source records attributed to R Einarsson.

At least 91 records · Page 5Linked to original sources
PubMed1980

Effects of the reduction and S-carbamidomethylation on the conformation, activity and heparin-binding capacity of human antithrombin III.

Antithrombin III has been shown to contain three disulphide bridges. They can be broken by reduction under non-denaturing conditions. The completely reduced and S-carbamidomethylated protein is devoid of thrombin-inhibiting activity and does not bind heparin. However, the immunological reactivity as determined by electroimmunoassay is almost unchanged, indicating that no large conformational changes occur upon cleavage of the disulphide bonds. This is also supported by the observation that the reduced and S-carbamidomethylated protein has the same fluorescence properties as the native protein dichroic spectrum of the native protein shows a pronounced pattern in the near-ultraviolet region, indicating restricted rotation of the aromatic amino acid side chains. This is drastically changed by the reduction and carbamidomethylation. The character of the far-ultraviolet circular dichroic spectrum is similar in the native and reduced S-carbamidomethylated protein. The reduction and S-carbamidomethylation of antithrombin III induces some local changes in the tertiary structure affecting the inhibitor activity, heparin binding and near-ultraviolet circular dichroic spectrum, but do not seem to induce any substantial general conformation change.

2,2'-Dipyridyl↗
PubMed1979

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernlöhr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.

Alcohol Oxidoreductases↗
PubMed1979

Characterization of highly purified native streptokinase and altered streptokinase after alkaline treatment.

Physical and chemical data are reported for highly purified native streptokinase (staphylokinase, EC 3.4.99.22) (Kabikinase) and streptokinase treated with an alkaline agent (altered streptokinase). The mol. wts. were similar and were determined to be 50 200 by sedimentation equilibrium methods, polyacrylamide gradient gel electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis. The sedimentation coefficient so20,w of native and altered streptokinase was found to be 3.37 S. The frictional ratio and the absorptivity (A1%1cm) at 280 nm of native streptokinase was found to be 1.29 and 7.5, respectively. Native streptokinase showed essentially a single band in the isoelectro-focusing pattern (pI 5.2), while altered streptokinase showed at least two separate bands. Polyacrylamide gel electrophoresis in the presence of Triton X-100 exhibited one band for native streptokinase but altered streptokinase showd two bands. At pH 12 the biological and immunological activity of streptokinase was markedly decreased in a time-dependent reaction. The amino-terminal amino acid of the two streptokinase forms was isoleucine and the carboxyl-terminal amino acid of native streptokinase was tyrosine. Peptide analysis showed that some peptides in altered streptokinase exhibited higher mobility compared to native streptokinase. The data suggest that streptokinase undergoes a conformational change when incubated in alkaline media, but no simultaneous loss of peptides was observed.

Amino Acids↗
PubMed1979

Use of electrophoresis and gel filtration to characterize human pituitary somatotropin preparations after storage.

Fresh and stored preparations of human pituitary somatotropin examined by different polyacrylamide gel electrophoresis techniques and by gel exclusion chromatography (Ultrogel) clearly demonstrated that the hormone undergoes a structural alteration during storage for 3 years. The formed aggregates were readily quantitated from the gel filtration profile and from polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). According to gel filtration the amount of aggregates in fresh and stored hormone was determined to be 2 and 10%, respectively, while SDS-PAGE indicated 6 and 14%.

Chromatography, Gel↗
PubMed1978

The increase in human antithrombin III tryptophan fluorescence produced by heparin.

The increase in fluorescence of human antithrombin III has been used to study the binding of a semi-synthetic heparin analogue. One molecule of heparin were found to bind with an association constant of 8.9 . 10(4) M-1. The intrinsic fluorescence of antithrombin III exhibits a fluorescence quantum yield of 0.17. Upon addition of heparin a marked increase in the protein fluorescence quantum yield is observed.

Antithrombins↗
PubMed1977

Binding of heparin to human antithrombin III as studied by measurements of tryptophan fluorescence.

Corrected fluorescence excitation and emission spectra of human antithrombin III have been determined. The fluorescence observed originates almost entirely from tryptophan residues. Reduction of the disulfide bonds followed by carboxymethylation did not change the fluorometric properties of the protein. The binding of heparin to antithrombin III caused a marked fluorescence enhancement by about 30% of the intrinsic protein emission intensity. Various samples of heparin yielded different binding curves. Heparin fractionated by gel filtration seemed to be bound to two sites on antithrombin III with association constants of 0.6-10(6)m-1 and 0.2-10(6)M-1 respectively. Heparin, prepared by affinity chromatography on matrix-bound antithrombin III appeared to be bound to only one site with an association constant of 2.3-10(6)M-1. Under similar conditions heparin caused no increase of the intrinsic protein emission intensity when added to reduced and carboxymethylated antithrombin III. The implications of these findings are discussed.

Alpha-Globulins↗
PubMed1977

Quenching of tryptophan fluorescence in human antithrombin III by iodide ion.

Iodide is an efficient quencher of antithrombin III intrinsic tryptophan fluorescence. The quenching pattern indicates that about 60% of the tryptophyl fluorescence originates from exposed residues in the multitryptophan-containing protein. In denaturing media all of the tryptophyls are solvent-exposed. The binding of heparin to antithrombin III influences the number of solvent-exposed tryptophan residues. By studying the dependence of the quenching on pH, information regarding the presence of charged residues adjacent to tryptophyls was obtained.

Antithrombins↗
PubMed1976

The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III.

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4-10(-5) M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3 - 10(-6) M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.

Anilino Naphthalenesulfonates↗
PubMed1975

Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies.

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.

Anions↗
PubMed1975

Catalysis of the photochemical dismutation of N-methylacridinium cation to N-methylacridone and N-methyl-9, 10-dihydroacridine by hydrophobic sites of horse-liver alcohol dehydrogenase and human serum albumin.

The N-methylacridinium cation is bound to hydrophobic sites of horse liver alcohol dehydrogenase and human serum albumin with an observed stoichiometry of one molecule N-methyl-acridinium chloride per subunit of alcohol dehydrogenase and 2.5 molecules of the dye per molecule human serum albumin; the dissociation constants are 3.6 X 10(-5) M and 1.7 X 10(-5) M, respectively. In light, the proteins catalyze the dismutation of N-methylacridinium chloride to N-methylacridone and N-methyl-9,10-dihydroacridine. The presence or absence of oxygen has no effect upon the observed reaction rate. If horse liver alcohol dehydrogenase is used as catalyst, the reaction is inhibited by adenosine diphosphoribose and by 1,1'-dimethyl-4,4'-bipyridylium dichloride. It is concluded that the N-methylacridinium cation is bound within the catalytic site of the enzyme interacting with the binding sites of the nicotinium ring and/or the binding site of the lipophilic part of the substrate. The anaerobic photodismutation of N-methylacridinium chloride to N-methyl-9,10-dihydroacridine and N-methylacridone can be explained by several alternative patways (see Appendix by S. Hünig), the overall reaction being 2[N-Methylacridinium]+ + H2Ohw leads to N-Methyl-9,10-dihydroacridine + N-methylacridone + 2H+. The prerequisite, a high rate of proton transfer from the reaction site, seems to be common property of the hydrophobic binding regions for the N-methylacridinium cation in both horse liver alcohol dehydrogenase and human serum albumin.

Acridines↗
PubMed1975

Binding of Au(CN)2- and Pt(CN)4-2- to horse liver alcohol dehydrogenase. A 35C1NMR relaxation study.

The binding of Au(CN)2- and Pt(CN)4-2- ions to the coenzyme binding site of horse liver alcohol dehydrogenase (alcohol : NAD+ oxidoreductase EC 1.1.1.1) has been studied by 35C1 nuclear magnetic relaxation. Longitudinal relaxation rates were analyzed in terms of a simple model and binding constants for Au(CN)2-, Pt(CN)4-2- and C1- were estimated. From a comparison between transverse and longitudinal relaxation rates the correlation time and the quadrupole coupling constant of bound chloride ion were obtained. The quadrupole coupling constant estimated from a simple electrostatic model for chloride ion interacting with an arginine group agrees with the experimental value.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

Animals↗