Search PubMed⌕ Search

Biomedical subjects

B Svensson

Publications and source records attributed to B Svensson.

At least 55 records · Page 3Linked to original sources
PubMed2000

Determination of the solution conformation of D-gluco-dihydroacarbose, a high-affinity inhibitor bound to glucoamylase by transferred NOE NMR spectroscopy.

The determination of the bound solution conformation of D-gluco-dihydroacarbose (GAC), a tight-binding inhibitor of several glycosidase and amylase enzymes, by glucoamylase is described. Transferred NOE NMR experiments and line-broadening effects indicate that GAC is bound in a conformation resembling that observed in the crystal structure. This contrasts with the predominant conformation of GAC when free in solution. The NMR results also suggest regions on the carbohydrate that are in close contact with the protein. The determination of the bound solution conformation of GAC by glucoamylase using transferred NOE (trNOE) measurements is a significant achievement given the high affinity constant (Ka = 3 x 10(7) M(-1)) for this receptor-ligand pair. It is striking that the off-rate for complexation is still sufficiently high to permit observation of trNOEs.

Carbohydrate Conformation↗
PubMed2000

Complexes of glucoamylase with maltoside heteroanalogues: bound ligand conformations by use of transferred NOE experiments and molecular modeling.

Transferred nuclear Overhauser effect (trNOE) experiments have been performed to investigate the conformations of the competitive inhibitors, methyl 5'-thio-4-N-alpha-maltoside 3a and methyl 5'-thio-4-S-alpha-maltoside 4 when bound to the catalytic subunit of the enzyme glucoamylase. These NMR data suggest that, although each of the free ligands populates two conformational families, both heteroanalogues are bound by the enzyme in conformations in the area of the global energy minimum. These conformations have been used as initial points for docking into the active site of the enzyme taken from a X-ray crystal structure of the related glucoamylase-D-gluco-dihydroacarbose 2 complex. Minimization of the resulting complexes has yielded structures for the bound complexes. Corroboration of the structures is provided by fast T(1)(rho)-relaxation effects for certain ligand protons as a result of close contacts with protons in the enzyme active site. The results auger well for the combined use of transferred NOE spectroscopy and molecular modeling based on X-ray crystal structures of complexes of suitable congeners for the rapid analysis of ligand-receptor interactions.

Acarbose↗
PubMed2000

Laser-induced shock wave endothelial cell injury.

BACKGROUND AND OBJECTIVE: Several laser procedures, extracorporeal lithotripsies (ESWL), and high-velocity missile trauma generate pressure transients that are transmitted through the tissues. Despite several publications demonstrating shock wave-induced tissue injury, little is known about its pathophysiology. This study introduces an in vitro model for studying shock wave effects on endothelial cell (EC) monolayers. STUDY DESIGN/MATERIALS AND METHODS: A Nd:YAG laser-driven flyer-plate technique was used to generate shock waves. Physical characteristics were determined with a pressure transducer, a high-speed video camera, and sequential photography. Biological effects were studied with phase contrast and lightfield microscopy, computerized morphometry, immunocytochemistry, spectrophotometry, and enzyme-linked immunosorbent assay (ELISA). RESULTS: The shock waves generated were highly reproducible. Cavitation was verified and quantified, and its extent could be varied in the vials. Exposed cultures exhibited areas with cell membrane damage and cell detachment. Release of LD was elevated (P < 0.01) in exposed vials. The EC lesions were larger (>P < 0.01) in cultures submitted to high vs. low extent of cavitation. CONCLUSION: The flyer-plate model can be used to subject cell monolayers to defined and reproducible shock waves causing immediate cell injury similar to the previously reported vascular lesions associated with ESWL, pulsed lasers, and blast trauma. With the flyer-plate model, such lesions may be further studied on the cellular and subcellular levels.

Aluminum Silicates↗
PubMed2000

Humoral immunity host factors in subjects with failing or successful titanium dental implants.

BACKGROUND: Treatment with titanium dental implants is in general successful. However, an unknown number of implants do not integrate and are removed either by exfoliation or at the time of second stage surgery. It would be of importance to identify subjects at risk and predict early implant failure. METHODS: In a retrospective study serum IgG antibody titers and avidity in sera from 40 subjects who had experienced titanium dental implant treatments with non-osseo-integration as the outcome (NOTI) and in sera from 40 age and gender matched control subjects who had received successful titanium dental implants (SOTI) were studied. Serum IgG titers to whole cell Actinomyces viscosus, Bacteroides forsythus, Porphyromonas gingivalis, Staphylococcus aureus, and Streptococcus intermedius sonicated antigen preparations were studied by ELISA. RESULTS: Serum IgG antibody titers to S. aureus were significantly higher in subjects with SOTI than in NOTI (p<0.001) suggesting that higher titers indicate protection against implant failure as a result of S. aureus infection. Statistically significant higher serum IgG antibody avidity to P. gingivalis and B. forsythus were found in subjects with SOTI than in subjects with NOTI (p<0.01 and p<0.001, respectively). Statistical analysis failed to demonstrate antibody titer or avidity differences to the other pathogens studied. The likelihood that SOTI was associated with a high OD reading for S. aureus was 13.1:1 (p<0.001). Whether subjects were edentulous or not, or if they had lost teeth because of periodontitis or caries did not seem to matter. CONCLUSION: Serum IgG antibodies relative to B. forsythus, P. gingivalis and S. aureus may be associated with the outcome of implant procedures and explain why early implant failures occur.

Adult↗
PubMed2000

Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation.

alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex.

Amino Acid Sequence↗
PubMed2000

Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization.

Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure. In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In alpha-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis-Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (beta/alpha)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which alpha1,3-linked glucosyl units are joined sequentially to maltose.

Enzyme Stability↗
PubMed2000

Antifilaggrin autoantibodies in early rheumatoid arthritis.

OBJECTIVE: To study antifilaggrin autoantibodies in patients with early rheumatoid arthritis. METHODS: Antifilaggrin autoantibodies were detected by immunoblotting (AFA) and by indirect immunofluorescence ('AKA') in sera from 112 patients with early RA. RESULTS: At baseline, 'AKA' were detected in 29% and AFA in 32% of sera, whereas after 12 months, they were present in 23% and 26% of sera, respectively. Significant associations between 'AKA', AFA, and rheumatoid factor were found. 'AKA' and AFA titres were significantly higher in patients with susceptibility alleles. No clear relation was observed between 'AKA', AFA, and disease activity but radiological progression tended to be more pronounced in patients having antifilaggrin autoantibodies. CONCLUSIONS: Antifilaggrin autoantibodies, being present in about one third of all new cases of RA, may have a value in early diagnosis. The present data suggest that these autoantibodies also may be a marker of disease severity.

Adult↗
PubMed2000

The efficacy of once weekly compared with two or three times weekly subcutaneous epoetin beta: results from a randomized controlled multicentre trial. Swedish Study Group.

BACKGROUND: Anaemia in haemodialysis patients can be effectively treated with erythropoietin. We investigated whether subcutaneous (SC) epoetin ss administered once weekly was as effective as the same weekly dosage given in two to three divided doses. METHODS: One hundred and fifty-eight patients (delivered Kt/V >1.0, where K=dialyser-renal urea clearance, t=dialysis time and V=filtration volume, obtained by urea kinetic modelling) were randomized to treatment with SC epoetin beta either once weekly (n=118), or to their original dosage two or three times weekly (control group, n=40) for 24 weeks. All patients received intravenous iron supplementation when necessary. RESULTS: Eight-eight patients in the once weekly group and 30 patients in the control group were treated for at least 16 weeks and are included in the analysis. Stable haemoglobin levels were maintained without epoetin dose increases in 73% of patients in both groups. Mean haemoglobin levels at randomization and after 16 and 24 weeks were 11.4, 11.1 and 11.1 g/dl, respectively, in the once weekly group compared with 11.2, 11.3 and 11.2 g/dl, respectively, in the control group. The mean weekly epoetin beta dosages at randomization and after 16 and 24 weeks were 102, 103 and 106 IU/kg bodyweight, respectively, in the once weekly group compared with 109, 109 and 115 IU/kg bodyweight, respectively, in the control group. No statistically significant between-group differences were apparent for changes in haemoglobin levels or epoetin beta dosages at week 24. CONCLUSIONS: Once weekly SC administration of epoetin beta is as safe and effective in maintaining haemoglobin levels in stable haemodialysis patients as two or three times weekly administration of the same total dose. By using the once weekly regimen, patients can avoid up to 104 injections per year. This would reduce clinic time for patients who do not self administer, and may also encourage self-administration and improve overall compliance.

Aged↗
PubMed2000

A clinical study of CPH 82 vs methotrexate in early rheumatoid arthritis.

OBJECTIVES: The objectives of this study were to evaluate the therapeutic efficacy of CPH 82 in comparison with methotrexate (MTX) in adult patients with early, active rheumatoid arthritis (RA) and to compare the tolerance and safety profiles of the two drugs. METHODS: The study was a 24-week, double-blind, randomized study in 10 centres of 100 patients with active RA, with a disease duration of less than 2 yr at the start of treatment, which consisted of either CPH 82 300 mg/day or MTX 10 mg/week. The six primary effect variables were: number of swollen joints, Ritchie's articular index, patient's pain score, patient's global score, Health Assessment Questionnaire (HAQ) and C-reactive protein (CRP). Erythrocyte sedimentation rate (ESR), physician's global score and the efficacy according to the European League Against Rheumatism (EULAR) and American College of Rheumatology (ACR) response criteria were also analysed. RESULTS: There was a significant improvement for both drugs in all variables. Significant differences between the drugs in favour of MTX were found only in patient's pain score, CRP and ESR. By the EULAR criteria, 76% and 86% were judged to be responders in the CPH 82 and MTX groups, respectively. By the ACR criteria, the corresponding figures were 58% and 64%. The most common side-effects were gastrointestinal, which were similar in both groups. The numbers of treatment failures due to adverse events were two with CPH 82 and 14 with MTX. CONCLUSIONS: The clinical effect of CPH 82 in this study was comparable to that of MTX at a dose of 10 mg/week. Both drugs reduced acute-phase reactants, MTX more effectively than CPH 82. The safety profile of CPH 82 was more favourable than that of MTX without folic acid supplementation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Remission and response to early treatment of RA assessed by the Disease Activity Score. BARFOT study group. Better Anti-rheumatic Farmacotherapy.

OBJECTIVE: To assess criteria for individual response and remission based on the Disease Activity Score (DAS) in patients with RA participating in a long-term observational study. METHODS: The EULAR (European League against Rheumatism) criteria for individual response and a recently proposed remission criterion, DAS < 1.6, were applied to 90 patients with RA after treatment for 2 yr with disease-modifying anti-rheumatic drugs (DMARDs) and/or corticosteroids. RESULTS: Seventy-six per cent of the patients were classified as responders (46% good and 30% moderate responders). Good responders had significantly more improvement in pain and Health Assessment Questionnaire (HAQ) score than moderate responders and non-responders. Paired comparisons showed significant X-ray progression both for moderate responders and non-responders but not for good responders. Twenty-nine per cent of all responders had an end-point DAS > 2.4, indicating active disease. In this group of responders, X-ray changes progressed significantly, but this could not be demonstrated in the group of responders with DAS < or = 2.4. Thirty-six per cent of the patients included in the study were classified as being in remission after 2 yr of treatment. The group of patients in remission showed no evidence of X-ray progression after 2 yr. CONCLUSIONS: Response and remission criteria based on DAS were useful in a study of patients with RA who were managed essentially as in clinical practice. The criteria used showed construct and criterion validity, although discriminant validity could not be shown. The application of valid criteria for response and remission in clinical practice may be a useful aid in the evaluation of treatment effects and in making treatment decisions for individual patients.

Arthritis, Rheumatoid↗
PubMed2000

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.

High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Amino Acid Sequence↗
PubMed2000

Validated multivariate models predicting the growth response to GH treatment in individual short children with a broad range in GH secretion capacities.

The aim of the study was to develop and validate models that could predict the growth responses to GH therapy of individual children. Models for prediction of the initial one and 2-y growth response were constructed from a cohort of 269 prepubertal children (Model group) with isolated GH deficiency or idiopathic short stature, using a nonlinear multivariate data fitting technique. Five sets of clinical information were used. The "Basic model" was created using auxological data from the year before the start of GH treatment and parental heights. In addition to Basic model data, the other four models included growth data from the first 2 y of life, or IGF-I, or GH secretion estimated during a provocation test (AITT) or a spontaneous GH secretion profile. The performance of the models was validated by calculating the differences between predicted and observed growth responses in 149 new GH treated children (Validation group) who fulfilled the inclusion criteria used in the original cohort. The SD of these differences (SD(res)) in the validation group was compared with the SD(res) for the model group. For the 1st y, the SD(res) for the Basic model was 0.28 SDscores. The lowest SD(res) (0.19 SDscores), giving the most narrow prediction interval, was achieved adding the 24h GH profile and data on growth from the first 2 y of life to the Basic model. The models presented permit estimation of GH responsiveness in children over a broad range in GH secretion, and with an accuracy of the models substantially better than when using maximal GH response during an provocation test. The predicted individual growth response, calculated using a computer program, can serve as a guide for evidence-based decisions when selecting children to GH treatment.

Adolescent↗
PubMed2000

Heparin induces synthesis and secretion of tissue factor pathway inhibitor from endothelial cells in vitro.

TFPI is a potent inhibitor of the extrinsic coagulation system constitutively synthesized by endothelial cells. A major portion of intravascular TFPI is stored associated with endothelial cells. and administration of unfractionated heparin (UFH) in vivo causes a prompt mobilization of TFPI into the circulation. The present study was conducted to investigate how UFH affected the synthesis, secretion and anticoagulant potency of TFPI in endothelial cells in vitro. A spontaneously transformed immortal endothelial cell line was used (ECV304). Stimulation of ECV304 cells with UFH caused a prompt dose-dependent (0-5 IU UFH/ml) release of TFPI to the medium accompanied by no change of TFPI at the surface membrane assessed by immunocytochemical methods. Northern blot analysis revealed two mRNA transcripts for TFPI with a molecular size of 1.4 kb and 4.4 kb, respectively. Stimulation of ECV304 cells for 24 hrs with various concentrations of UFH caused a dose-dependent increase of TFPI in the medium (6.2-29.6 ng/10(6) cells within the concentration range 0-10 IU/ml). A similar dose-dependent increase in the expression of both TFPI mRNA species was observed. Long-term incubation of ECV304 cells with 5.0 IU/ml UFH caused a 5-10 fold increase in the TFPI concentration accumulated in the medium over 48 hrs. The increased TFPI mRNA expression induced by UFH appeared already after 10 min, peaked after 2-4 hrs, remained augmented throughout the entire period of UFH exposure, and preceded the synthesis-dependent increase in TFPI release by 2-4 hrs. The procoagulant activity of the cells was downregulated by 36% and the contribution of TFPI to the anticoagulant potency of ECV304 cells was moderately increased after 24 hrs heparin stimulation. It is suggested that these mechanisms are of major importance for the anticoagulant function of heparins.

Antibodies↗
PubMed2000

Temporomandibular disorders in juvenile chronic arthritis patients. A clinical study.

In a prospective study, 105 children with juvenile chronic arthritis (JCA) were clinically and radiographically examined. The aim was to investigate the distribution of symptoms and clinical signs of temporomandibular disorders and to study correlations with radiographic mandibular condylar lesions. The present material appeared to be a representative sample of Swedish JCA children with respect to the distribution of genders and JCA subtypes as well as the peak of onset of the disease. Symptoms from the masticatory system were common (26%) and pain at mandibular function and stiffness at mouth opening the most frequent ones. Restricted maximal voluntary mouth opening (MVM) was the most frequent clinical finding. Radiographic condylar lesions, frequently found in the present material (39%), were significantly correlated with postnormal occlusion, restricted MVM, anterior open bite and mandibular retrognathia. However, most of the JCA children with radiographic condylar lesions did not present with symptoms from the masticatory system, postnormal occlusion, restricted MVM, anterior open bite or mandibular retrognathia.

Adolescent↗
PubMed1999

Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase.

The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.

Binding Sites↗
PubMed1999

alpha-1,4-glucan lyases producing 1,5-anhydro-D-fructose from starch and glycogen have sequence similarity to alpha-glucosidases.

In the past few years a novel enzyme alpha-1,4-glucan lyase (EC 4.2. 2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known alpha-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75-80% among the alpha-1, 4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal alpha-1,4-glucan lyases is only 25-28%. Notably database searches disclosed that the alpha-1,4-glucan lyases have a clear identity of 23-28% with alpha-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and alpha-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in alpha-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of alpha-1,4-glucan lyases with alpha-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley.

The gene encoding the starch debranching enzyme limit dextrinase, LD, from barley (Hordeum vulgare), was isolated from a genomic phage library using a barley cDNA clone as probe. The gene encodes a protein of 904 amino acid residues with a calculated molecular mass of 98.6 kDa. This is in agreement with a value of 105 kDa estimated by SDS-PAGE. The coding sequence is interrupted by 26 introns varying in length from 93 bp to 825 bp. The 27 exons vary in length from 53 bp to 197 bp. Southern blot analysis shows that the limit dextrinase gene is present as a single copy in the barley genome. Gene expression is high during germination and the steady state transcription level reaches a maximum at day 5 of germination. The deduced amino acid sequence corresponds to the protein sequence of limit dextrinase purified from germinating malt, as determined by automated N-terminal sequencing of tryptic fragments coupled with matrix assisted laser desorption mass spectrometry. The sequenced peptide fragments cover 70% of the entire protein sequence, which shows 62% and 77% identity to that of starch debranching enzymes from spinach and rice and 37% identity to Klebsiella pullulanase. Sequence alignment supports the multidomain architecture and identifies both secondary structure elements of the catalytic (beta/alpha)8-barrel substrate, catalytic residues, and specificity associated motifs characteristic of members of the glycoside hydrolase family 13 which cleave alpha-1,6-glucosidic bonds. A remarkable distribution of the secondary structure elements to individual exons is observed.

Amino Acid Sequence↗