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T Korpela

Publications and source records attributed to T Korpela.

At least 55 records · Page 3Linked to original sources
PubMed1993

Structural similarity of the binding sites of cyclophilin A-cyclosporin A and FKBP-FK506 systems.

Two enzymes, cyclophilin A and FK506-binding protein, with similar cis-trans isomerization catalytic activities but no similarity on the amino acid sequence level were compared in their three-dimensional structure by computer modelling. Cyclophilin A and FK506-binding protein proved to have similar arrangements at nine of the amino acids at their active site pockets. Two inhibitory peptides, cyclosporin A and FK506, also have structural similarities at their contact regions to the active sites. The studied systems may be another example of convergent evolution of enzyme catalytic site.

Amino Acid Isomerases↗
PubMed1993

Purification, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp. alkalophilus.

An intracellular beta-glucosidase was purified from cell extracts of Bacillus circulans subsp. alkalophilus by NAD affinity and high-performance anion-exchange chromatographies. The enzyme was active against a wide range of aryl-beta-glucosides and beta-linked disaccharides. The structural gene for beta-glucosidase was cloned in Escherichia coli. The beta-glucosidase gene consisted of an open reading frame of 1,350 bp encoding a protein of 450 amino acids with a calculated M(r) of 51,303. The enzyme exhibited from 45 to 66% identity with five bacterial beta-glucosidases.

Amino Acid Sequence↗
PubMed1993

Separate contributions of large- and small-scale dynamics to the heat capacity of proteins. A new viscosity approach.

We present here a theoretical description and experimental verification of a novel method of separating large-scale (LS) and small-scale (SS) dynamics contributions to the heat capacities of proteins in solution by using viscosity dependences. It was assumed that the LS dynamics, related to the thermal fluctuations of domains and subunits, is dependent on solvent viscosity within a sucrose concentration of 0-15% w/v, in contrast with the SS dynamics, which are related to fluctuations of atoms and amino acid residues. The results obtained with immunoglobulin G, hemoglobin and cytochrome c were in reasonable accordance with the previous data achieved by a spin-label method: the LS dynamics decrease, whereas the SS dynamics increase within the temperature elevation. The changes are dependent on the stage of the active sites of the proteins. The internal compensation of molecular dynamics between the LS and SS fluctuations may be an important factor in the design of thermostable enzymes and proteins. A new phenomenon concerning the ability of macromolecules to increase free volume in water as a result of ice-like clusters stabilization is revealed.

Hot Temperature↗
PubMed1993

Pyrophosphatase-based enzyme-linked immunosorbent assay of total IgE in serum.

Human IgE was purified to near homogeneity by a two-step procedure consisting of immunoaffinity chromatography and high-performance liquid chromatography. Mouse hybridoma cell lines secreting antibodies against IgE were generated. Monoclonal and polyclonal antibodies were used to develop a "sandwich"-type ELISA for determining total IgE in human serum. Inorganic pyrophosphatase (EC 3.6.1.1), an enzyme having a high turnover number, was used as the label. The mean analytical recovery of our ELISA was 95.2% and the results showed good linear correlation with an established RIA of IgE. We found pyrophosphatase to be a good alternative label for use in immunoassays.

Animals↗
PubMed1992

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.

Cysteine↗
PubMed1992

Characterization of bacterial growth on solid medium with image analysis.

Alkaliphilic bacterial strains producing the enzyme cyclodextrin glucanotransferase were cultivated on solid agar medium containing an indicator system detecting the enzyme. The growth of the colony and the surrounding diffusion zone, due to the enzyme, were measured by the image analysis during the cultivation. It was possible to differentiate between relatively similar clones by observing quantitatively the changes at and around the colony. Optimal experimental conditions for such measurements are discussed. The image analysis technique provides a potential tool for characterizing microbes grown on solid media.

Bacteria↗
PubMed1990

Analysis of cyclomaltodextrin glucanotransferase isoenzymes by isoelectric focusing in immobilized pH gradients.

The catalytically active subforms of cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19) from Bacillus circulans var. alkalophilus and from a strain in which the CGTase expressing gene had been cloned were studied by using isoelectric focusing (IEF) in immobilized and in conventional pH gradients. Even with high protein loads the best resolution was achieved in immobilized pH gradients (IPG). Native CGTase, focused on IPG 4.5-5.4, was resolved into more than 6 subforms, a major one with pI 4.97 and the others between pH 4.75 and 4.99. Cloned CGTase focused on the same IPG gel was resolved into more than 7 subforms over the pH range 4.78-5.22, with one major component of pI 5.20. A sensitive assay for the detection of CGTase on analytical gel slices was developed. The different behaviour between native and cloned CGTases was most probably a consequence of the presence--in the latter--of four extra amino acids at the N-terminus. A study of the reaction products by the enzymatic subforms confirmed that they are catalytically identical, hence small differences outside the active site are responsible for the presence microheterogeneity of the protein.

Amino Acid Sequence↗
PubMed1990

Flow-injection analysis of amino acids and their metabolites by immobilized vitamin B6-dependent enzymes. Sensitive determination of L-aspartate, L-glutamate, 2-oxoglutarate, and oxaloacetate.

Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed. The analytes were enzymatically coupled with NADH which was monitored by light emission from immobilized bacterial bioluminescence enzymes. Aspartate (or oxaloacetate) was assayed on the basis of NADH consumption by introducing the sample through a coimmobilized aspartate aminotransferase-malate dehydrogenase column. The assay responded linearly from 100 pmoles to 5 nmoles per assay. Glutamate (2-oxoglutarate) was determined by formation of NADH in the glutamate dehydrogenase reaction. The measuring range for glutamate was from 10 pmoles to 100 nmoles per assay. The precision of the flow-injection method was generally excellent, and the sensitivities of the described assays were 100-1000-fold higher than with spectrophotometric methods. The immobilized enzyme preparations were stable for several months in storage, and the enzyme columns could be used for 600-800 analyses. Flow-injection analyses of amino acids and related compounds by NADH/bioluminescence-coupled reactions provide a sensitive, fast, and inexpensive assay method for a wide variety of purposes.

Animals↗
PubMed1989

Immobilization of aspartate aminotransferase on agarose.

Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.

Aldehydes↗
PubMed1988

Acid-base chemistry of vitamin B6 compounds in methanol.

The enzymatic reactions involving pyridoxal 5'-phosphate (PLP) can be simulated in solutions; thus, this system forms a favorable model for understanding the requirements of the enzymatic catalysis. We have studied in methanol protonic equilibria of the imines formed between PLP or salicylaldehyde (SA) and various amino acids, using UV and NMR spectroscopy. A glass electrode and an operational pH* scale were used to control acidity. The first protonation of the phosphate of PLP imines can be detected by UV spectroscopy with pK* at 10.8, proved by [31P]-NMR. The second protonation of phosphate (pK* at 4.8) is accompanied by increased hydrolysis of the imines. The imines of aspartate deviate from the imines of nondicarboxylic amino acids indicating that the beta-carboxyl of aspartate is internally hydrogen-bonded. PLP-2-aminobutanol Schiff base does not show with [1H]-NMR at pH* 7 separate peaks for ketoenamine-enolimine tautomers even at -90 degrees C, SA-phenylalanine shows an unidentified absorption at 350-380 nm. This was tentatively assigned a trans structure.

Acid-Base Equilibrium↗
PubMed1987

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol.

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.

Aminooxyacetic Acid↗
PubMed1987

Colorimetric determination of beta-cyclodextrin: two assay modifications based on molecular complexation of phenolphtalein.

The decolorization of phenolphtalein upon complexation to cyclodextrins was studied to measure beta-cyclodextrin concentrations. Several factors possibly affecting the self-life of the dye were tested. By making the assays in 0.1 M NaCO3 solution beta-cyclodextrin concentrations down to 6 microM (SNR = 2) could be determined while the practical assay range reached up to 0.06 mM. In this form the method was unaffected by acyclic oligosaccharides and directly applicable to cyclodextrin assays in complex starch hydrolyzates. The method was further modified to be used in a flow-injection analyzator and the results were comparable to those obtained by HPLC analyses after prepurification of the samples.

Autoanalysis↗
PubMed1986

Alpha 1-antitrypsin in acute anterior uveitis and rheumatic diseases.

To test the pathogenetic role of the phenotype MZ of alpha 1-antitrypsin/alpha 1-protease inhibitor (PI) in acute anterior uveitis (AAU) and in different rheumatic diseases we examined 360 unrelated patients including 93 with AAU alone, 24 patients with AAU and ankylosing spondylitis (AS), 21 patients with AAU and Reiter's disease (RD), 26 patients with AAU, AS, and RD 54 patients with AS alone, 16 patients with RD alone, 115 patients with rheumatoid arthritis (RA) alone, and 11 patients with psoriatic arthritis (PA) alone. Of the 164 AAU patients, 80 had a single attack, and 84 had repeated episodes. There were neither significant differences between different groups of the patients and 120 healthy controls nor between patients with AAU alone and patients with AAU and AS or RD in the frequencies of the PI phenotypes tested. The results indicate that the PI MZ type is not closely associated with AAU, AS, RD, RA and PA and that it does not play any role in determining whether AAU shows a pattern of a single attack or repeated episodes, and whether AAU occurs alone or together with AS or RD.

Acute Disease↗
PubMed1984

Multiple forms of aspartate aminotransferase as studied by liquid chromatography.

A convenient method, based on high-performance Liquid chromatography (HPLC) is described for the analysis of multiple forms of cytosolic aspartate aminotransferase. The method has a better resolution; in the same time it is drastically more rapid than the conventional chromatography on CM-Sephadex. The results suggest that transition forms arising from the heat denaturation of the enzyme can be detected by HPLC.

Animals↗
PubMed1984

Equilibria between vitamin B6-compounds and cyclodextrins.

Convenient chromatographic methods to measure equilibrium constants between cyclodextrins and aromatic compounds were developed. The use of 'Hummel-Dreyer' elution profile allows us to determine values above 15-20 M-1. The weaker associations (below 15-20 M-1) can be measured with cyclo-dextrin-derivatized Biogel P-2 column. The latter method is very suitable to determine the equilibrium constants between cyclodextrins and vitamin B6-compounds which can't be measured with the conventional photometric procedure.

Chemical Phenomena↗
PubMed1983

Comparison of the coupling recoveries of immobilized aspartate aminotransferase. Specific activity, enzyme-bound coenzyme, and transaminationable active centers.

Aspartate aminotransferase (AspAT, EC 2.6.1.1) was bound on CNBr-activated Sepharose and the effects of immobilization on the maximum velocity, biologically active pyridoxal-5'-phosphate (PLP), and transaminationable active centers were studied. By comparing these parameters of soluble and immobilized enzyme the factors decreasing the observed reaction rate upon immobilization were evaluated. Ninety percent of the soluble protein in the coupling mixture was bound to the support. The amount of enzyme-bound PLP of immobilized preparation was 83% of that of the soluble one. The coupling recovery of specific activity was 46%, which was 10%-units lower than that of the transaminationable active centers. This difference depends on the fact that a part of the active centers of immobilized enzyme had lower catalytic rate, due to the enzyme-matrix interactions or internal mass transfer limitations, than the others. The immobilized catalytically active AspAT had 80% of the turnover efficiency of the soluble enzyme. The affinity of the enzyme to its substrates did not significantly change upon immobilization, neither did the pH profile.

Aspartate Aminotransferases↗