Salivary Α-amylases Research Articles

Complete amino acid sequence of an α-amylase inhibitor in wheat kernel (0.19-inhibitor)

Amino acid composition of the 0.19-inhibitor from wheat kernel is very similar to that of the 0.53-inhibitor, but a marked difference in inhibitory activity towards human salivary and pancreatic α-amylases was detected between the two inhibitors. Elucidation of the primary structure of the 0.19-inhibitor and structural comparison with the 0.53-inhibitor is essential to understand not only the mechanism of the selective inhibitory behaviors but also evolutional relationship of these inhibitors. The complete amino acid sequence of the 0.19-inhibitor was determined after cleaving the protein with cyanogen bromide and trypsin. As in the case for the 0.53-inhibitor, the 0.19-inhibitor is composed of two identical subunits with 124 amino acid residues. Comparison of the sequence of the 0.53- and 0.19-inhibitor shows very high sequence homology with amino acid substitutions at seven positions.

PURIFICATION AND PARTIAL CHARACTERIZATION OF TWO ?-AMYLASE INHIBITORS FROM BLACK BEAN (Phaseolus vulgaris)

Two α-amylase inhibitors from black bean (Phaseolus vulgaris) were purified to homogencity using ammonium sulfate fractionation, DEAE-Sephadex chromatography, phenyl-Sepharose hydrophobic interaction chromatography and gel filtration with Sephadex G-100. The inhibitors were designated I–1 and I–2 based on their order of elution from the phenyl-Sepharose column. Both inhibitors are mannose containing glycoproteins, composed of subunits; active against porcine pancreatic, human salivary, and insect α-amylases and inactive against bacterial, mold, and plant α-amylases. The inhibitors I-1 and I–2 have molecular weights of 49,000 and 47,000 and isoelectric points 4.93 and 4.86, respectively. Both inhibitors have similar amino acid compositions and are rich in aspartic acid, serine, glutamic acid, valine, and threonine and are low in sulfur containing amino acids. I–2 is more resistant to heat denaturation than I-1.

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous. Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.

Differential assay of human pancreatic and salivary α-amylases in serum using a new fluorogenic substrate

The difference in the mode action of human pancreatic and salivary α-amylases on O-6- deoxy-6-[(2- pyridyl)amino]-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)- d-glucitol (FG6R), a fluorogenic derivative of maltohexaitol, was found. The products of the enzymatic hydrolysis were analyzed by high-performance liquid chromatography (HPLC) in 8 min. FG6R was hydrolyzed by these enzymes to O-6- deoxy-6-[(2- pyridyl)amino]-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)- d-glucose (FG3) and maltotriitol, or O-6- deoxy-6-[(2- pyridyl)amino]-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)-O-α- d-glucopyranosyl -(1 → 4)- d-glucose (FG4) and maltitol. Pancreatic α-amylase produced more FG4 than salivary α-amylase. Taking advantage of the differences in action of the two amylases, a differential α-amylase assay in serum was performed. The method is simple and rapid and can be used for routine clinical assays of α-amylases.

Kinetics of human pancreatic and salivary α-amylases with carboxymethylamyloses as substrates

The enzymatic measurement of α-amylase in human serum with chemically modified amylose as substrate was studied. Several substitutions of carboxymethylamylose were prepared from amylose, suitable amounts of monochloroacetic acid, and sodium hydroxide. The relationship between the degree of substitution of the Carboxymethylamylose and the kinetic parameters of human pancreatic, salivary and porcine pancreatic α-amylases was determined. The action of α-amylases on such modified amyloses in the presence of glucoamylase was measured by a specific enzymatic assay having glucose as the product. The kinetic parameters of human pancreatic α-amylase were similar to those of human salivary α-amylase. It was possible to determine suitable sensitivities for the α-amylase assay using this substrate. These results suggested a subsite model.

Enzyme digestion in the proximal digestive tract of the pig: A review

Studies on enzyme digestion in the precaecal part of the digestive tract show that the pig posesses digestive enzymes that can completely break down dietary substrates to absorbable nutrients. Gastric pepsins, pancreatic proteolytic enzymes and intestinal peptidases are responsible for the hydrolysis of dietary proteins; salivary and pancreatic α-amylases start the hydrolysis of starch, its products being broken down by intestinal carbohydrases; dietary lipids, mainly triglycerides, are hydrolyzed in the intestinal lumen by the complex lipase—colipase—bile salts. Hydrolysis of food components may be complete, depending upon different factors such as the composition of the diet or the nature of the substrate.

Kinetic difference between hydrolyses of γ-cyclodextrin by human salivary and pancreatic α-amylases

γ-Cyclodextrin was found to be hydrolyzed by human salivary and pancreatic α-amylases (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) at appreciable rates. The optimum pH for the enzyme reactions at 37°C in the presence of 0.1 M NaCl was at around pH 5, which was remarkably different from the optimum pH (pH 6.9) of the enzymes for starch. The K m value (2.9 mg/ml) of pancreatic α-amylase for γ-cyclodextrin was smaller than that (5.3 mg/ml) of salivary α-amylase at pH 5.3, while the V value of the former was 3.7-times larger than that of the latter. The hydrolyses of γ-cyclodextrin by both enzymes took place via the multiple attack mechanism. The degrees of multiple attack by salivary and pancreatic α-amylases for γ-cyclodextrin at pH 5.3 were 2.0 and 1.1, respectively. The distribution of maltodextrins produced by hydrolysis of γ-cyclodextrin by salivary α-amylase was suggested to be independent of the substrate concentration, while that produced by pancreatic α-amylase was presumably dependent on the substrate concentration.

Interaction of human α-amylases with inhibitors from wheat flour

The interaction of four purified α-amylase (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic α-amylases was investigated. The inhibitory activity of the four proteins towards salivary α-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic α-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic α-amylase and inhibitor 4 was observed, though complex formation between pancreatic α-amylase and the other inhibitors was not clearly demonstrated.

Isolation and characterization of four inhibitors from wheat flour which display differential inhibition specificities for human salivary and human pancreatic α-amylases

Four α-amylase (1,4-α- d-glucan glucanohydrolase, EC 3.2.1.1) inhibitors were isolated from an albumin fraction of wheat flour by ion-exchange and gel-filtration chromatography. The purified inhibitors were characterized according to their electrophoretic mobilities, molecular weights, carbohydrate, content, sulphydryl content, susceptibility to proteolytic digestion and specificities in inhibiting human salivary and pancreatic α-amylases. The properties of these inhibitors are compared to similar proteins isolated by other workers.

Action of human pancreatic and salivary α-amylases on maltooligosaccharides: Evaluation of kinetic parameters

The kinetic studies on the reactions of human pancreatic and salivary α-amylases with several maltooligosaccharides (maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were carried out. The susceptibility to hydrolysis with human pancreatic α-amylase decreased in the order of maltopentaose, maltohexaose, maltotetraose, and maltoheptaose, while with human salivary α-amylase maltopentaose was hydrolysed slightly slower than maltohexaose but fairly faster than maltotetraose or maltoheptaose from a viewpoint of the rates of reactions based on the amount of substrate changed. The relative rates of production of substrates, utilized in the coupled yeast α-glucosidase reaction, increased in the order of maltoheptaose, maltohexaose, maltotetraose, and maltopentaose with human pancreatic α-amylase, while with human salivary α-amylase in the order of maltoheptaose, maltotetraose, maltohexaose, and maltopentaose. Thus, maltopentaose was considered to be the best substrate over maltotetraose, maltohexaose or maltoheptaose for the α-glucosidase coupled method of α-amylase determination.

Activity of serum α-amylases in cystic fibrosis

A new method which uses a differential inhibitor to measure pancreatic and salivary type α-amylases (EC 3.2.1.1) was applied to serum samples from 46 cystic fibrosis (CF) patients (age range 4–14 years) and 50 controls of the same age group. The levels of pancreatic type amylase were lower in the CF patients (median 26.5 I.U./l) than the controls (median 81.5) ( P < 0.001). The results for salivary-type enzyme, however, did not support the previously reported finding of higher than usual levels in CF patients. This discrepancy is probably due to differences in analytical methods. It is felt that this procedure will be of value in the investigation of patients for cystic fibrosis and other pancreatic disorders.

PURIFICATION AND CHARACTERIZATION OF ?-AMYLASE INHIBITORS IN WHEAT (TRITICUM AESTIVUM VAR. ANZA)

Three α-amylase inhibitors were purified to homogeneity from Anza wheat (Triticum aestivum var. Anza) by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE-cellulose, CM-ceillulose and Sephadex G-50. Homogeneity was determined by disc gel and isoelectric focusing electrophoresis and by sedimentation equilibrium centrifugation. The inhibitors are designated 0.19, 0.28 and 0.55 on basis of their relative electrophoretic mobilities on polyacrylamide gels. The 0.19, 0.28 and 0.55 inhibitors had molecular weights of 24,000, 18,500 and 30,000 by polyacrylamide gel electrophoresis with different gel concentrations while the former two were 29,000 and 14,500 by sedimentation equilibrium centrifugation, respectively. The molecular weight of the 0.55 inhibitor was not determined by centrifugation. The isoelectric points were 5.9, 5.2 and 4.2 for the 0.19, 0.28 and 0.55 inhibitors, respectively. The three inhibitors had similar amino acid compositions but differed significantly in amounts of lysine, arginine, histidine, alanine, valine and phenylalanine. The 0.19 inhibitor was active against human salivary and hog pancreatic α-amylases but inactive against Bacillus subtilis and Aspergillus oryzae α-amylases. The 0.28 inhibitor had very weak activity against only human salivary α-amylase. The 0.55 inhibitor had activity against only human salivary α-amylase. The 0.55 inhibitor appears to differ from all previously reported wheat α-amylase inhibitors while the 0.28 inhibitor (protein) is unique in having essentially no inhibitory activity.

PURIFICATION AND CHARACTERIZATION OF AN ?-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.