Human Amylase Research Articles

Interpretation of polymorphic DNA patterns in the human α-amylase multigene family

Previous molecular studies have clearly shown that the human amylase locus has a very complicated structure. Multiple salivary and pancreatic amylase genes are present on haplotypes with variable numbers of genes. To study the population heterogeneity, human genomic DNA from family members and random individuals was digested with a number of different restriction enzymes and hybridized with probes representing various parts of the human pancreatic amylase cDNA. The complex patterns obtained were, in most cases, compatible with predictions from the restriction enzyme maps of cloned human amylase genes. With some enzymes deviations from the predicted intensities of the bands associated with the pancreatic amylase gene AMY2A were observed. These findings can be explained by unequal homologous crossovers between AMY2A and AMY1A, resulting in haplotypes with one gene less or one gene more than the haplotypes described thus far. Moreover, a very complicated TaqI polymorphism was found that can be explained by homologous crossovers between different salivary amylase genes. Because some salivary amylase genes have an inverted orientation with respect to the others, these data provide evidence for the occurrence of intrachromosomal, homologous crossovers, as proposed by us previously (P. C. Groot et al., 1990, Genomics 8: 97–105).

Effects of synthetic human pancreastatin on pancreatic secretion and blood flow in rats and dogs

Effects of synthetic human pancreastatin-52 and human pancreastatin-29 on pancreatic secretion and blood flow were examined in rats and dogs. Synthetic human pancreastatin-52 and human pancreastatin-29 were equally potent in suppressing the release of amylase stimulated by cholecystokinin in rats in vivo. However, neither human pancreastatin-52 nor human pancreastatin-29 altered basal and cholecystokinin-stimulated amylase release from isolated dispersed rat pancreatic acini. In studies in dogs, human pancreastatin-29 suppressed releases of amylase and protein stimulated by cholecystokinin, but did not alter pancreatic blood flow. These results suggest that the inhibitory effects of pancreastatin on pancreatic secretion do not involve a direct action on pancreatic acinar cells nor alteration of pancreatic blood flow. Pancreastatin probably is important in regulating exocrine pancreatic secretions as well as endocrine pancreatic secretions.

Evolution of the human α-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1–3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.

The effect of resistant starch on colon function in humans

Starch that is resistant to human amylases forms during the cooking and subsequent cooling of some foods, and may therefore be a substrate for the bacterial flora of the colon. It is thus possible that resistant starch (RS) will affect colon function in a similar manner to non-starch polysaccharides. To test this theory, a group of eight volunteers took two diet supplements for 1 week each in a random order with a 1 week separation. One supplement comprised mainly 350 g Cornflakes/d and the other 380 g Rice Krispies/d, providing 10.33 and 0.86 g RS/d respectively. The amounts of amylase-digestible starch, nonstarch polysaccharides, total carbohydrate, energy, protein and fat were balanced between the two periods by giving small amounts of Casilan, wheat bran, butter and boiled sweets. The volunteers made faecal collections during day 3 to day 7 of each period. Whole-gut transit time was calculated using the continuous method. Stool consistency and ease of defaecation were assessed by the volunteers. All episodes of flatulence noticed were recorded in a diary, along with food intake. Serial breath hydrogen measurements were made at 15 min intervals for 8 h on day 1 of each supplement. Questionnaires regarding colon function were completed at the end of each dietary period. There were no significant differences in the stool mass, frequency or consistency, ease of defaecations, transit time or flatulence experienced during the two supplements (P greater than 0.05). Significantly more H2 (area under curve) was produced while eating Cornflakes than Rice Krispies (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic synthesis of p-nitrophenyl 3 5- O- β- N-acetylglucosaminyl|-α-maltopentaoside by lysozyme; a novel substrate for human amylase assay

Transglycosylation from di- N-acetylchitobiose to the 3-position at the nonreducing end glucosyl group of p-nitrophenyl α-maltopentaoside was regioselectively induced through the use of hen egg-white lysozome. The enzyme formed p-nitrophenyl 3 5- O- β- N-acetylglucosaminyl- α-maltopentaoside (5% of the enzyme-catalyzed net decreased of p-nitrophenyl α-maltopentaoside) from di- N-acetylchitobiose as a donor and p-nitrophenyl α-maltopentaoside as an acceptor. The rate of the transglycosylation depended on the concentration of substrate, the temperature and the pH. The hydrolytic actions of human pancreatic and salivary α-amylase on this derivative were examined. The maltopentaoside derivative was shown to be useful as a substrate for α-amylase assay through a coupled reaction involving α- D-glucosidase and glucoamylase.

Retroviral and pseudogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution.

We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.

Retroviral and Pseudogene Insertion Sites Reveal the Lineage of Human Salivary and Pancreatic Amylase Genes from a Single Gene during Primate Evolution

We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.

Mapping the human amylase gene cluster on the proximal short arm of chromosome 1 using a highly informative (CA) n repeat

The human amylase gene cluster includes a (CA) n repeat sequence immediately upstream of the γ-actin pseudogene associated with the AMY2B gene. Analysis of this (CA) n repeat by PCR amplification of genomic DNA from the 40 families of the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel revealed extensive polymorphism. A total of six alleles with (CA) n lengths of 16–21 repeats were found. The average heterozygosity for this polymorphism was 0.70. Multipoint linkage analysis showed that the amylase gene cluster is located distal to the nerve growth factor β-subunit gene (NGFB) and is within 1 cM of the anonymous locus D1S10. The amylase (CA) n repeat provides a convenient marker for both the physical and the genetic maps of human chromosome 1p.

Identification of a novel α-amylase by expression of a newly cloned human amy3 cDNA in yeast

A novel amylase gene ( amy3) that differs in nucleotide sequence from salivary amylase gene ( amyl) and pancreatic amylase gene ( amy2) has been described [Tomita et al., Gene 76 (1989) 11–18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel α-amylase gene ( amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.

Enzymatic synthesis of p-nitrophenyl .ALPHA.-maltoheptaoside by transglycosylation of maltohexaose-forming amylase.

An extracellular maltohexaose-forming amylase [EC 3.2.1.98] from Klebsiella pneumoniae mutant is a normal hydrolytic enzyme that hydrolyzes short-chain amylose (DP = 23) to give about 40% maltohexaose. Transglycosylation from maltoheptaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltohexaose-forming amylase in an aqueous methanol solution. The enzyme specifically produced p-nitrophenyl alpha-maltoheptaoside (13% of the p-nitrophenyl alpha-glucoside) from maltoheptaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The yield of p-nitrophenyl alpha-maltoheptaoside depended on the concentration of methanol solvent, the pH, and temperature. Furthermore, the use of the aqueous methanol system in the reaction not only improved the solubility of p-nitrophenyl alpha-glucoside but also greatly increased the formation of p-nitrophenyl alpha-maltoheptaoside, which is a useful substrate for assay of human amylase in serum and urine.

Radioimmunoassay of Human Pancreatic Amylase with Monoclonal Antibody

A monoclonal antibody (E-21) was obtained that specifically binds to human pancreatic amylase and shows negligible cross-reaction with human salivary amylase. Using this antibody a radioimmunoassay was developed for pancreatic amylase in human serum. The assay was shown to be sensitive (detectable up to 7 mg/L), reproducible, and specific for pancreatic amylase. In normal subjects, the mean concentration of serum pancreatic amylase determined by this method was 36.3 mg/L with a 95% confidence range of 16.5 to 79.2 mg/L. A good correlation was observed between the concentrations of immunoreactive pancreatic amylase (IR-PA) and enzymatic activities in 20 serum samples (r = 0.97). The concentration of serum IR-PA was below the detectable limit in pancreatectomised patients, and was greatly increased in patients with acute pancreatitis; the latter was accompanied by parallel changes in total enzymic activity. In patients with mumps, the serum IR-PA level was within the normal range whereas the total enzymic activity was elevated.

Relationship of Urinary Amylase Activity and Proteinuria in the Dog

-I In normal dogs and in some dogs with marked hyperamylasemia there is very little urinary amylase a c t i ~ i t y . ' . ~ . ~ . ~ . ~ ~ This is surprising since the molecular weight of purified canine pancreatic amylase determined using a denaturing technique is about 54,000 daltons) which is below the threshold of the glomerular filter. It has been hypothesized that canine amylase does pass the glomerular filter but is totally reabsorbed from the filtrate or is ina~tivated.~ Recently, it has been shown that these mechanisms probably do not play an important role in amylase clearance and that canine amylase probably does not pass the intact glomerular filter.6 It appears that unique characteristics of the canine glomerular filter and amylase might account for the lack of appearance of urinary amylase activity. It has been reported that native human amylase appears as a polymer.8 Since the polymerization phenomenon may be important in understanding the clearance of canine amylase, an estimate of the molecular weight of canine serum amylase and the influence of this weight on glomerular filtration were determined. Four pools of canine sera, each composed of ten canine sera chosen randomly from routine laboratory accessions at the University of Guelph Veterinary Teaching Hospital, were made. Random urine samples were obtained from 13 normoamylasemic 1-year-old Samoyed dogs in a colony bred to propagate the sex-linked trait for hereditary glomerulopathy.' Urine samples were obtained from 16 normoamylasemic, non-hematuric, non-hemoglobinuric dogs with proteinuria. Total amylase activities were determined using a p-nitrophenylmaltohexaoside substrate (Coulter Electronics of Canada, Ltd., Burlington, Ontario, Canada). Urinary protein concentrations were determined by a Ponceau S (Aldrich Chemical Co., Milwaukee, WI) dye binding method and purified human albumin standards (Dade Diagnostics, Inc., Aguada, Puerto R i ~ o ) . ~ Urinary specific gravities were determined refractometrically. The four pools of canine sera were each diluted 300 times in a buffer containing 0.086 M NaC1, 7 mM KCl, 2 mM MgCl,, 7 mM CaCI,, and 0.02 M tris(hydroxymethy1)-aminomethane-HC1 (pH 7.2). Aliquots of diluted pooled sera were concentrated to the original volume of serum using 100 and 300 kilodalton molecular weight cut-off ultrafilters (Amicon Canada Ltd., Oakville, Ontario, Canada). Prior to use, 10 ml of a 0.15 M NaCl solution containing l.Oo/o bovine serum albumin was passed through each ultrafilter to prevent non-specific adherence. The ultrafiltration was done at 4 C and 10 Ib/sq. in. About 20% of albumin is retained by both sizes of ultrafilters (Amicon Canada Ltd., Oakville, Ontario, Canada). Amylase activities in the retentates were determined and results expressed as the percent amylase activity retained by each filter. The relations between urinary protein concentration, specific gravity, and amylase activity were determined by linear --

Aspergillus oryzae has two nearly identical Taka-amylase genes, each containing eight introns

cDNA and genomic DNA for two nearly identical genes, AmyI and AmyII, coding for the enzyme Takaamylase A (TA-A) of the fungus Aspergillus oryzae have been cloned and characterized. These genes are apparently unlinked, differing by only 3 nucleotides (nt) out of the 2720 nt that span the coding regions. The 617-nt 5'-flanking regions differ only at nt −372 (T or A) from the putative ATG start codon and contain four sets of short, inverted repeats (IR) upstream from the putative TATAAA box at nt -100 and the transcription start point at nt −69. The coding regions consist of 499 codons disrupted by eight intervening sequences. The putative proenzymes differ by only two amino acids (aa) and consist of the 478-aa extracellular enzyme plus a 21-aa hydrophobic leader sequence. Except for the replacement site changes in codons 35 (Arg a3 Gln) and 151 (Phe a3 Leu), the identity of the two genes continues downstream for 58 nt past the TG A stop codon before diverging. Exon 9 codes for 94 of the 98 aa of the domain B of mature TA-A. Little conservation of TA-A exons was found when these exons were aligned with those of human amylase. The genes are flanked by at least 6 to 10 kb of unrelated chromosomal nucleotide sequence. The Amy genes are co-expressed, since mRNA (cDNA) specific to the 3 ' -UTR of both genes was recovered from mycelia grown on starch, a known inducer of TA-A biosynthesis. The 3'-UTRs of cDNAs related to AmyI are shorter (128 nt and 145 nt) than those of AmyII (179 nt and 297 nt). The AmyI specific 3'-UTR is characterized by the absence of IR sequences and the presence of a putative ‘AATAAA’ polyadenylation signal.

High amount of dietary carbohydrate does not cause an adaptational increase of stimulated human pancreatic amylase secretion

In order to investigate whether the human pancreas is capable of adapting to a diet with high-carbohydrate, low-fat, and normal protein content, 10 healthy volunteers were given a defined elemental diet (60% of calories as carbohydrates, 22% as fat, and 18% as protein) for 7 d. For the next 7 d they received an elemental diet with a further increased carbohydrate content (76% of calories) and a decreased fat content (10% of calories). A complete secretin-pancreozymin test was carried out at the end of the first wk and at d 14. The results show that an increase in dietary carbohydrate does not provoke an adaptational response of stimulated secretion rates of amylase, trypsin, and chymotrypsin in humans, as expected from animal experiments.

In vitro Digestibility of Cereal and Legume (Phaseolus vulgaris) Starches by Bovine, Porcine and Human Pancreatic α‐Amylases Effect of Dietary Fiber

AbstractThe effect of heat treatment and dietary fiber from whole and dehulled black bean (Phaseolus vulgaris) seeds on digestibility of various starches by bovine, porcine and human pancreatic α‐amylases was studied. Corn, rice, wheat and black bean starch digestibility increased after heating. The degree of hydrolysis of black bean starch ranged between 56 and 83% of the values obtained for cereal starches depending on the enzyme used. Enzyme activity was: human‐ > porcine > bovine regardless of the substrate employed. Dietary fiber decreased starch digestibility by porcine enzyme. This effect was more marked when whole grain fiber was used. Black bean and rice starch digestibility by human amylase was not affected by fiber, while corn and wheat starch hydrolysis was slightly inhibited. The use of the human enzyme is recomended for in vitro amylolysis assays. Attention is called on the difficulties of extrapolating results obtained with animal enzymes to humans.

市販抗ｌｉｇｈｔ鎖抗体液中に含まれるヒトアミラーゼ失活因子

市販抗ｌｉｇｈｔ鎖抗体液中に含まれるヒトアミラーゼ失活因子

Enzymatic Synthesis of p-Nitrophenyl α-Maltopentaoside in an Aqueous-Methanol Solvent System by Maltotetraose-Forming Amylase: A Substrate for Human Amylase in Serum

Transglycosylation from maltopentaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltotetraose-forming amylase from Pseudomonas stutzeri in an aqueous solution containing methanol at a high concentration. The enzyme specifically formed p-nitrophenyl alpha-maltopentaoside (12% of the enzyme-catalyzed net decrease of maltopentaose) from maltopentaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The rate of the transglycosylation depended on the concentration of methanol solvent, the pH and the temperature. Use of the aqueous methanol system in this reaction not only ensured a sufficient solubility of p-nitrophenyl alpha-glucoside but also resulted in a remarkable increase in the formation of p-nitrophenyl alpha-maltopentaoside, which is a useful substrate for assay of human amylase in serum and urine.

Concerted Evolution of Human Amylase Genes

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.

Concerted evolution of human amylase genes.

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.

Ｌｅｖａｎｓｕｃｒａｓｅによるヘテロフルクトオリゴ糖の合成

1. Bacillus subtilis levansucrase was isolated from cultured cells grown on a medium containing sucrose at 37°C for 40 hr and purified as an electrophoretically pure state which is free from invertase.2. The enzyme produced levan predominantly toward sucrose alone. But heterofructooligosaccharides were synthesized under the co-existence of mono or oligosaccharide. The maximum efficiency (80%) of transfructosylation from sucrose to acceptor sugars by the enzyme obtained in the case of the acceptor/donor=l or more in molar ratio.3. Oligosaccharide formed by the levansucrase of Bacillus subtilis from sucrose and reducing oligosaccharide was non reducing sugar that C2 of fructosyl residue is linked to Cl of reducing end of acceptor sugars.4. Levansucrase linked to CH-sepharose 4B to produce immobilized enzyme was increased thermal stability 30°C to 40t and used repeatedly to make maltooligosaccharide-F without loss of the activity.5. The susceptibility of oligosaccharide-F to human amylases was tested. Heterooligosaccharides produced by levansucrase can be used as a substrate for screening of new carbohydrolases.