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B Svensson

Publications and source records attributed to B Svensson.

At least 181 records · Page 10Linked to original sources
PubMed1992

Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.

Aspergillus niger↗
PubMed1992

Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates.

Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes.

Barwin is a basic protein with pI above 10 and molecular mass 13.7 kDa isolated from aqueous extracts of barley seed. The complete amino acid sequence of 125 residues has been determined by a combination of conventional protein sequencing, plasma desorption mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Three disulfide bridges have been localized as Cys31-Cys63, Cys52-Cys86, and Cys66-Cys123 both by 1H nuclear magnetic resonance spectroscopy and by plasma desorption mass spectrometry. The N-terminal residue was identified as pyroglutamate. Barwin is closely related to a peptide segment of 122 residues at the C-terminal region of the proteins encoded by two wound-induced genes in potato plants, win1 and win2, and a protein encoded by the hevein gene of rubber tree. In 77 sequence positions of 125 the barwin, win1, win2, and hevein protein sequences have amino acid sequence identity, when two gaps--one of two residues allowing for the insert of Gly23 and Ala24 and one allowing for the insert of Thr97 in the barwin sequence--are introduced in the latter three. The close sequence similarity with the proteins encoded by the wound-induced potato and rubber tree genes and the ability of the protein to bind saccharides suggest that barwin might belong to a group of proteins involved in a common defense mechanism in plants.

Amino Acid Sequence↗
PubMed1992

Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates.

The specificity constants, kcat/KM, were determined for glucose oxidase and glucose dehydrogenase using deoxy-D-glucose derivatives and for glucoamylase using deoxy-D-maltose derivatives as substrates. Transition-state interactions between the substrate intermediates and the enzymes were characterized by the observed kcat/Km values and found to be very similar. The binding energy contributions of individual sugar hydroxyl groups in the enzyme/substrate complexes were calculated using the relationship delta(delta G) = -RT ln [(kcat/KM)deoxy/(kcat/KM)hydroxyl] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4- and 6-OH groups (4'- and 6'-OH in maltose), where changes in binding energies from 10 to 18 kJ/mol suggested strong hydrogen bonds between the enzymes and these substrate OH groups. The 3-OH (3'-OH in maltose) was involved in weaker interactions, while the 2-OH (2'-OH in maltose) had a very small if any role in transition-state binding. The three enzyme-substrate transition-state interactions were compared using linear free energy relationships (Withers, S. G., & Rupitz, K. (1990) Biochemistry 29, 6405-6409) in which the set of kcat/KM values obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (rho) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis.

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by difference spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojirimycins, (c) D-glucono-1,5-lactone, and (d) maltitol, two types of disaccharide substrates (a) alpha-(1----4)-linked and (b) alpha-(1----6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with the valienamine-derived inhibitors or when D-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116----Ala and Trp120----Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (M) were determined: acarbose, less than 6 x 10(-12); methyl acarviosinide, 1.6 x 10(-6); and the D-gluco and L-ido forms of hydrogenated acarbose, 1.4 x 10(-8) and 5.2 x 10(-6), respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophanyl residues, although each had a Kd value of approximately 4 x 10(-6) M. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a starch-granule-binding site in the C-terminal domain. The alpha-, beta-, and gamma-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

Aspergillus niger↗
PubMed1992

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex.

Molecular recognition and site-directed mutagenesis are used in combination to identify kinetically, transition state interactions between glucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180----Gln GA had indicated a role in substrate binding for Glu180 (Sierks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193-198). Here, changes in activation energies calculated from measured kcat/Km values for a series of deoxygenated maltose analogues indicate hydrogen bonding between the mutant enzyme and the 3-OH group of the reducing end sugar ring. Using the same substrate analogues and determining activation energies with wild-type GA an additional hydrogen bond with the 2-OH group of maltose is attributed to an interaction with the carboxylate Glu180. This novel combination of molecular recognition and site-directed mutagenesis enables an enzyme substrate transition state contact to be identified and characterized even without access to the three dimensional structure of the enzyme. Given the distant structural relationships between glucoamylases and several starch hydrolases (Svensson,B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activity of these related enzymes.

Binding Sites↗
PubMed1991

Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity.

The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates.

Amylases↗
PubMed1991

Comparison of the domain-level organization of starch hydrolases and related enzymes.

Structure-prediction and hydrophobic-cluster analysis of several starch hydrolases and related enzymes indicated the organization of eleven domain types. Most enzymes possess a catalytic (beta/alpha)8-barrel and a smaller C-terminal domain as seen in crystal structures of alpha-amylase and cyclodextrin glucanotransferase. Some also have a starch-granule-binding domain. Enzymes breaking or forming endo-alpha-1,6 linkages contain domains N-terminal to the (beta/alpha)8-barrel.

Amino Acid Sequence↗
PubMed1991

C-terminal processing of barley alpha-amylase 1 in malt, aleurone protoplasts, and yeast.

C-terminal processing of low pI barley alpha-amylase (AMY1) results in multiple forms in malt, aleurone protoplasts, and transformed yeast. Expression of an AMY1 cDNA in yeast thus leads to four secreted forms with distinct pI values between 4.7 and 5.1 and essentially identical Mr. AMY1-1 and AMY1-2 lacking the C-terminal Arg-Ser are generated by carboxypeptidase in vitro from AMY1-3 and AMY1-4, respectively. In vivo processing is due to the KEX1-encoded yeast carboxypeptidase. AMY1-2 and AMY1-4 are fully active, whereas AMY+-1 and AMY1-3 retain 3-4% activity toward p-nitrophenyl maltoheptaoside and have one fewer SH group, due to reaction with glutathione. AMY1-1-AMY1-4 are indistinguishable from malt AMY1 with respect to Ca(2+)-, substrate-, and beta-cyclodextrin-binding as well as recognition by three monoclonal antibodies and limited proteolysis by proteinase K. Transient AMY1 precursors present in barley aleurone protoplasts were trapped by addition of serine carboxypeptidase inhibitors, indicating that endogenous carboxypeptidase participates in the maturation of AMY1 during germination. Three pairs of precursor/mature AMY1 forms are recognized, presumably corresponding to the three genes encoding AMY1. Malt carboxypeptidase II can convert in vitro the precursors isolated from protoplasts into processed enzyme, and AMY1 from malt accordingly lacks the C-terminal heptapeptide. This report thus demonstrates posttranslational protein modification by carboxypeptidase in higher plants.

Amino Acid Sequence↗
PubMed1991

Ion-binding properties of calbindin D9k: a Monte Carlo simulation study.

Monte Carlo simulations are used to calculate the binding constant of two Ca2+ ions to the protein bovine calbindin D9k. The change in binding constant with respect to mutation of charged amino acids, presence of various electrolytes, protein concentration, solution pH, and competitive binding of monovalent ions is investigated. Each of these factors may have a large influence on the binding constant. The simulations are performed in a dielectric continuum model, the so-called primitive model of electrolyte theory, with a fixed protein structure and a uniform dielectric permittivity. The calculated binding constants are in excellent agreement with experimental data and describe changes in the binding constant over six orders of magnitude.

Animals↗
PubMed1991

Electrostatic interactions in protein solution--a comparison between Poisson-Boltzmann and Monte Carlo calculations.

The accuracy of the Poisson-Boltzmann (PB) approximation and its linearized version is investigated by comparison to results obtained from Monte Carlo simulations. The dependence of the calcium binding constant of the protein calbindin as a function of salt concentration and mutation is used as a test case. The protein is modeled as a collection of charged and neutral spheres immersed in the electrolyte solution. The PB equation is solved using a finite difference technique on a grid in a spherical polar coordinate system, which is the preferred choice for a globular protein like calbindin. Both MC and PB give quantitative agreement with experimental results. The linearized PB equation is almost as accurate, but it becomes less reliable in systems with divalent ions. However, the linearized PB equation fails to describe the concentration profiles for cations and anions outside the protein even in a 1:1 salt solution.

Anions↗
PubMed1991

European EVA decompression sickness risks.

For the first manned flight of Hermes there will be a capability of performing EVA. The European EVA Space Suit will be an anthropomorphic system with an internal pressure of 500 hPa of pure oxygen. The pressure reduction from the Hermes cabin pressure of 1013 hPa will induce a risk for Decompression Sickness (DCS) for the EVA crewmember if no adequate protective procedures are implemented. Specific decompression procedures have to be developed. From a critical review of the literature and by using knowledge gained from research conducted in the past in the fields of diving and aerospace medicine safe protective procedures are proposed for the European EVA scenario. An R factor of 1.2 and a tissue half-time (t1/2) of 360 minutes in a single-tissue model have been identified as appropriate operational values. On the basis of an acceptable risk level of approximately 1%, oxygen prebreathing times are proposed for (a) direct pressure reduction from 1013 hPa to a suit pressure of 500 hPa, and (b) staged decompression using a 700 hPa intermediate stage in the spacecraft cabin. In addition, factors which influence individual susceptibility to DCS are identified. Recommendations are also given in the areas of crew selection and medical monitoring requirements together with therapeutic measures that can be implemented in the Hermes scenario. A method for demonstration of the validity of proposed risks and procedures is proposed.

Aerospace Medicine↗
PubMed1991

Serum enriched with n-3 polyunsaturated fatty acids inhibits procoagulant activity in endothelial cells.

Nine healthy male volunteers (mean age 24.3 years, range 21-27 years) ingested 25 ml cod liver oil (CLO) daily for 8 weeks. Blood samples were collected before and after the intervention period. A slight increase in total serum cholesterol (+ 12.2%, P less than 0.01) and high density lipoprotein cholesterol (+ 16.9%, P less than 0.01) were calculated to have a non-significant effect on the high/low density lipoprotein ratio. Polyunsaturated fatty acids in serum were displaced from the n-6 family to the n-3 family, reflected by an eight-fold increase of the eicosapentaenoic (20:5 n-3)/arachidonic (20:4n-6) acid ratio. Human umbilical vein endothelial cells were grown in a medium to which 30% sterile filtered serum, collected before and after CLO supplementation for 72 h, was added. After 2 h exposure to lipopolysaccharides the thromboplastin activity in endothelial cultures incubated with serum enriched with n-3 fatty acids was 43% lower than in cultures containing serum collected before the intervention (1.86 +/- 0.48 10(-3)/10(6) cells vs 3.26 +/- 0.85 10(-3)/10(6) cells). It is suggested that serum prepared from subjects given n-3 fatty acids may support the thromboresistence of endothelial cells.

Adult↗
PubMed1990

Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins.

Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗