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PubMed1987

Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall.

We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.

Antibodies, Monoclonal↗
PubMed1976

Serum amylase determinations and amylase to creatinine clearance ratios in patients with chronic renal insufficiency.

Patients with severe chronic renal failure may have significant hyperamylasemia in the absence of clinical symptoms or signs of acute pancreatitis. Amylase to creatinine clearance (CA/CC) ratios were usually elevated in patients with chronic renal failure and were not helpful in evaluating the possibility of acute pancreatitis. The mean amylase to creatinine clearance ratio for the controls with normal renal function was 1.24 +/- 0.13. In patients with chronic renal failure, it was 3.17 +/- 0.42 (P less than 0.001). Serum amylase isoenzyme patterns revealed no difference in salivary to pancreatic isoenzyme ratios between normals (1.04 +/- 0.12) and patients with severe renal insufficiency without evidence of pancreatic disease (1.07 +/- 0.13). The isoenzymes were helpful in excluding the diagnosis of pancreatic in 1 renal failure patient whose hyperamylasemia was primarily salivary in origin and in confirming the diagnosis in another who had only a pancreatic band.

Adult↗
PubMed1982

The genetic linkage between the PKU locus and the loci for amylase 1, amylase 2, Fy, PGM 1, and Rh and the question of assignment of the PKU locus to chromosome no. 1.

The alpha-amylase loci Amy 1 and Amy 2 and other loci on chromosome 1 were investigated for their linkage relationship to the PKU locus. Ten families were informative for the study of linkage between PKU/Amy, 20 for PKU-Fy, 11 for PKU/PGM 1, and 10 for PKU/Rh linkage. The probabilities of linkage at different recombinant fractions were calculated according to Bayes' theorem. The results are in striking contrast with those of Kamaryt et al. who found strong evidence for close linkage between the amylase loci and the PKU locus, whereas with our results close linkage can be excluded; loose linkage is possible but unlikely. The results are discussed with regard to the genetic heterogeneity of phenylketonuria.

Amylases↗
PubMed1978

Studies on the substrate specificity of Taka-amylase A1. XIV. Preparation of 6-deoxy-6-halogenomaltotrioses and their hydrolysis by Taka-amylase A.

1. O-6-Deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-chloro-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-bromo-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated. 2. The Michaelis constant Km and the molecular activity ko were determined at 37 degrees C and pH 5.2 using the modified maltotrioses. The values of Km and ko decreased upon modification of maltotriose and those of ko/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl alpha-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate. 3. All halogenomaltotrioses examined showed substrate inhibition at high substrate concentrations.

Amylases↗
PubMed1982

Studies on heterogeneity of Taka-amylase A: isolation of an amylase having one N-acetylglucosamine residue as the sugar chain.

Crystalline Taka-amylase A, prepared from Takadiastase, was fractionated into four fractions by DEAE-Sephacel and Concanavalin A-Sepharose column chromatography. The relative weight ratio of the fractions was 90 : 4 : 4 : 2. They had similar molecular weights (51,000), amino acid compositions, and hydrolytic activity against soluble starch, but different phenyl maltosidase activities and electrophoretic mobilities on polyacrylamide gel electrophoresis. Three of the fractions mainly had the high mannose type sugar chain with the sugar composition of Man5-GlcNAc2, but the other fraction had only one N-acetylglucosamine residue as the sugar chain. These results suggested that Taka-amylase A was heterogeneous both in the sugar portions and in the polypeptide portions.

Acetylglucosamine↗
PubMed1983

High amylase content of neoplastic pleural and pericardial effusion probably secondary to amylase producing tumor cells: report of 2 cases.

We report two cases of malignant pleural and pericardial effusion respectively secondary to bronchogenic carcinomas. In both effusions, a significant elevation of the Salivary-type-amylase fraction was found, while the corresponding values were normal in serum and urine. Electronmicroscopy of the malignant tumor cells from the pleural effusion showed typical electron-dense granules, suggesting zymogen granules. It is concluded that the high amylase content of the effusions was due to secretion of S-type-isoamylase by the tumor cells.

Aged↗
PubMed1991

Modification of alpha-amylase from Bacillus licheniformis by the polyaldehyde derived from beta-cyclodextrine and alpha-amylase thermostability.

The cleavage of beta-cyclodextrine by sodium periodate at the seven 2-3 diols of the glucose unit gives rise to the polyaldehyde 1, used to modify alpha-amylase. The reductive modification of alpha-amylase from Bacillus licheniformis reduced the number of reactive lysine groups from 8 to 3.5 per mol of enzyme with an activity loss of 25% and increased the half-life at 80 degrees C from 4.7 to 7.0 minutes.

Aldehydes↗
PubMed1995

Stopped-flow kinetic studies of the reaction of barley alpha-amylase/subtilisin inhibitor and the high pI barley alpha-amylase.

The interaction of alpha-amylase/subtilisin inhibitor (BASI) from barley seeds and the high pI barley alpha-amylase (AMY2) de novo synthesized during seed germination, has been studied at pH 8.0, 25 degrees C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: [formula: see text] The resulting values of the kinetic parameters were: k2/K1 = (1.0 +/- 0.2) x 10(6) M-1.s-1, K1 = 0.4 +/- 0.21 mM, k2 = 320 +/- 150 s-1, k-2 = (7.2 +/- 0.6) x 10(-5)s-1, and the overall dissociation constant Kd = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2.

Hordeum↗
PubMed1994

The active center of a mammalian alpha-amylase. Structure of the complex of a pancreatic alpha-amylase with a carbohydrate inhibitor refined to 2.2-A resolution.

An X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (EC 3.2.1.1) that was soaked with acarbose (a pseudotetrasaccharide alpha-amylase inhibitor) showed electron density corresponding to five fully occupied subsites in the active site. The crystal structure was refined to an R-factor of 15.3%, with a root mean square deviation in bond distances of 0.015 A. The model includes all 496 residues of the enzyme, one calcium ion, one chloride ion, 393 water molecules, and five bound sugar rings. The pseudodisaccharide acarviosine that is the essential structural unit responsible for the activity of all inhibitors of the acarbose type was located at the catalytic center. The carboxylic oxygens of the catalytically competent residues Glu233 and Asp300 form hydrogen bonds with the "glycosidic" NH group of the acarviosine group. The third residue of the catalytic triad Asp197 is located on the opposite side of the inhibitor binding cleft with one of its carbonyl oxygens at a 3.3-A distance from the anomeric carbon C-1 of the inhibitor center. Binding of inhibitor induces structural changes at the active site of the enzyme. A loop region between residues 304 and 309 moves in toward the bound saccharide, the resulting maximal mainchain movement being 5 A for His305. The side chain of residue Asp300 rotates upon inhibitor binding and makes strong van der Waals contacts with the imidazole ring of His299. Four histidine residues (His101, His201, His299, and His305) are found to be hydrogen-bonded with the inhibitor. Many protein-inhibitor hydrogen bond interactions are observed in the complex structure, as is clear hydrophobic stacking of aromatic residues with the inhibitor surface. The chloride activator ion and structural calcium ion are hydrogen-bonded via their ligands and water molecules to the catalytic residues.

Acarbose↗
PubMed2003

Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry.

The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.

Calcium↗
PubMed1999

Phage-displayed peptide ligands for pancreatic alpha-amylase cross-react with barley alpha-amylase.

Peptide ligands that bind to pancreatic alpha-amylase were isolated from bacteriophage libraries displaying random 15-mer peptides by iterative affinity selection and amplification. The DNA sequences of selected clones from the final round of biopanning were determined. The two phage-display ligands with high-binding activities contained a high content of Arg, Tyr, and Trp residues with the short consensus sequence Arg-X-Tyr-Trp. These clones were shown to exhibit comparable binding interactions toward barley alpha-amylase based on transducing units titering and measurement of the dissociation constants.

Amino Acid Sequence↗
PubMed1987

Characterization of alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum. Evidence for a novel thermostable amylase.

Thermostable extracellular alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum E 101-69 were characterized in a crude enzyme preparation. The activities responded similarly to temperature and pH, with optima at 85-90 degrees C and pH 5.6. The activities were stable at 65 degrees C, but were inactivated gradually in an identical manner at higher temperatures in the absence of Ca2+ and substrate. Ca2+ stabilized both activities similarly at high temperatures. Ca2+ also stimulated both activities, whereas EDTA reversed this stimulation. The activities were similarly inactivated at pH extremes. The two activities distributed in the same way during isoelectric focusing. The results suggest that the two activities are properties of the same protein, representing a novel, thermostable, amylase.

Amylose↗
PubMed1995

Arg-27, Arg-127 and Arg-155 in the beta-trefoil protein barley alpha-amylase/subtilisin inhibitor are interface residues in the complex with barley alpha-amylase 2.

Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents. Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2-complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI. Since Arg-27 is not conserved in rice and wheat ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155].

Amino Acid Sequence↗
PubMed2004

Inhibitory effect of 0.19 alpha-amylase inhibitor from wheat kernel on the activity of porcine pancreas alpha-amylase and its thermal stability.

The inhibitory effect of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel on the porcine pancreas alpha-amylase (PPA)-catalyzed hydrolysis of p-nitrophenyl-alpha-D-maltoside (pNP-G2) was examined. 0.19 AI is a homodimer of 26.6 kDa with 13.3-kDa subunits under the conditions used. The elution behaviors in gel filtration HPLC of PPA and 0.19 AI indicated that a PPA molecule bound with a 0.19 AI molecule (homodimer) at a molar ratio of 1:1. 0.19 AI inhibited PPA activity in a competitive manner with an inhibitor constant, K(i), of 57.3 nM at pH 6.9, 30 degrees C, and the binding between them was found to be endothermic and entropy-driven. The activation energy for the thermal inactivation of 0.19 AI was determined to be 87.0 kJ/mol, and the temperature, T(50), giving 50% inactivation in a 30-min incubation at pH 6.9 was 88.1 degrees C. The high inhibitory activity of 0.19 AI against PPA and its high thermal stability suggest its potential for use in the prevention and therapy of obesity and diabetes.

Animals↗
PubMed2006

Comparison of the wild-type alpha-amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal stability, and insights into engineering the thermal stability of bacillus alpha-amylase.

The starch hydrolysis activity and thermal stability of Bacillus amyloliquefaciens alpha-amylase (wild-type enzyme or WT) and its variant enzymes, designated as M77, M111, and 21B, were compared. All have an optimal pH at around 6, as well as almost the same reaction rates and Km and kcat values. The optimal temperature in the absence of Ca2+ ions is 60 degrees C for WT and M77 and 40 degrees C for M111 and 21B. Those of M111 and 21B rose to 50-60 degrees C upon the addition of 5 mM CaCl2, while those of WT and M77 did not change. The dissociation constants Kd for Ca2+ to WT and M77 are much lower than those of M111 and 21B. Asp233 in WT is replaced by Asn in M111 and 21B, while it is retained in M77, suggesting that Asp233 is involved in the thermal stability of the enzyme through Ca2+ ion binding. These findings provide insight into engineering the thermal stability of B. amyloliquefaciens alpha-amylase, which would be useful for its applications in the baking industry and in glucose manufacturing.

Amino Acid Sequence↗
PubMed2006

Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis alpha-amylases and insights into engineering alpha-amylase variants active under acidic conditions.

Bacillus licheniformis alpha-amylase (BLA) is widely used in various procedures of starch degradation in the food industry, and a BLA species with improved activity at higher temperature and under acidic conditions is desirable. Two BLA species, designated as PA and MA, have been isolated from the wild-type B. licheniformis strain and a mutant strain, respectively. In this study, their starch-hydrolysis activity and thermal stability were examined. MA showed higher activity than PA, especially at acidic pH (pH 5.0-5.5), and even after 1 h of treatment at 90 degrees C. MA was active in the range of pH 4.0-8.0, which is much wider than that (pH 4.5-7.5) of PA. It was shown that the proton dissociation constants on the acidic and alkaline sides (pKa1 and pKa2) were shifted to more acidic and basic values, respectively, by the mutation of PA to MA. The activation energy and thermodynamic parameters for their thermal inactivation indicate that MA is more thermally stable and catalytically active than PA, suggesting that MA could be useful for glucose-production process coupled with reactions catalyzed by beta-amylase.

Amino Acid Sequence↗
PubMed1996

Complete sequence, subunit structure, and complexes with pancreatic alpha-amylase of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

The complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, alpha and beta, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (alpha beta)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic alpha-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA.

Amino Acid Sequence↗
PubMed1997

Structural characterization of an alpha-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous alpha-amylase inhibitors.

The primary structures of two subunits of an alpha-amylase inhibitor (alpha AI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previously deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of alpha AI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that alpha AI-2 has the subunit stoichiometry of an alpha 2 beta 2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) alpha-amylase inhibitor, which is quite different in the inhibitory specificity from alpha AI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.

Amino Acid Sequence↗