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At least 109 records · Page 6Linked to original sources
PubMed1989

Interaction of immobilized anti-salivary amylase antibody with human macroamylases: implications for use in a pancreatic amylase assay to distinguish macroamylasemia from acute pancreatitis.

We examined the ability of an immobilized antibody to salivary amylase (Clin Chem 1985;33:1283-8) to react with amylase in macroamylasemic sera. The antibody removed 50% (SD 23%) of the total amylase activity from 39 macroamylase sera, a percentage indistinguishable (P greater than 0.75) from the percentage removed from concurrently analyzed sera from healthy volunteers (49%, SD 11%). Electrophoretic analysis of 23 macroamylasemic sera revealed that the antibody removed only part of the macroamylase band(s) in 71% of the cases. We conclude that the mean isoenzyme composition of the macroamylase complexes is essentially identical to the mean isoenzyme distribution in normal sera (i.e., about half salivary and half pancreatic amylase). Further, the immobilized antibody can be used to distinguish most patients with macroamylasemia from those with acute pancreatitis, because sera from the latter contain an increased proportion (greater than 80%) of pancreatic amylase.

Acute Disease↗
PubMed1995

Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor.

We developed an automated method for measurement of alpha-amylase isoenzymes in serum by a single kinetic assay (SKA) and a double kinetic assay (DKA) with 2-chloro-4-nitrophenyl-6(5)-azido-6(5)-deoxy-beta-maltopentaoside as a substrate and 6(3)-deoxymaltotriose (DOG3) as a novel selective amylase inhibitor. DOG3 showed a large difference in inhibitory activity between human pancreatic alpha-amylase (HPA; 86.9% inhibition) and salivary alpha-amylase (32.1% inhibition) at 0.33 mmol/L. Constant inhibition was obtained immediately after addition of DOG3. The inhibitory effect did not change with variation in concentrations of amylase up to approximately 3000 U/L. The results obtained by SKA correlated well with those obtained by three methods: monoclonal antibody (r = 0.988), wheat germ inhibitor (r = 0.989), and DKA (r = 0.995). The within-run and between-run CVs for HPA were 0.63-2.32% on SKA, 0.69-1.81% on DKA. No significant interferences by endogenous serum compounds were observed with the proposed methods.

Autoanalysis↗
PubMed1977

Influence of amylase assay technique on renal clearance of amylase-creatinine ratio.

The influence of amylase assay technique on the renal amylase/creatinine clearance measurement was determined by analysis of serum and urine specimens obtained from 10 normal subjects. CAm/CCr averaged 2.19 +/- 0.18% with a saccharogenic technique, 1.52 +/- 0.2% with an iodometric technique, and 0.80 +/- 0.08% with a chromogenic technique. Each of these values differed significantly (P less than 0.05) from the other two. Recovery studies were carried out by adding partially purified human salivary or pancreatic amylase to human newborn serum or urine (which contain minimal endogenous amylase). Equal amylase activity was recovered from serum and urine by the saccharogenic technique whereas recovery from urine was less than 50% of that from serum using the iodometric and chromogenic techniques. The accuracy of the chromogenic technique is markedly improved by the addition of albumin to the urine assay system. Although it appears that only the saccharogenic method provides an accurate estimate of CAm/CCr, each assay technique distinguished the elevated CAm/CCr of patients with pancreatitis from the normal range established for that technique. Accurate clinical interpretation of CAm/CCr measurment requires knowledge of the amylase assay technique used.

Amylases↗
PubMed1994

Parallel beta/alpha-barrels of alpha-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase versus the barrel of beta-amylase: evolutionary distance is a reflection of unrelated sequences.

The structures of functionally related beta/alpha-barrel starch hydrolases, alpha-amylase, beta-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, are discussed, their mutual sequence similarities being emphasized. Since these enzymes (except for beta-amylase) along with the predicted set of more than ten beta/alpha-barrels from the alpha-amylase enzyme superfamily fulfil the criteria characteristic of the products of divergent evolution, their unrooted distance tree is presented.

Amino Acid Sequence↗
PubMed1981

Action pattern of human pancreatic alpha-amylase on maltoheptaose, a substrate for determining alpha-amylase in serum.

An enzymatic assay for the determination of alpha-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of alpha-glucosidase and the hexokinase-glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of alpha-amylase, (B) that of the combined action of alpha-amylase and alpha-glucosidase. Conductive to this effect was: the availability of pure maltoheptaose and human pancreatic alpha-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchange) and of an HPLC system for separation of substrate and reaction products without interference from by products of the assay (partition chromatography on a cation-exchange column with acetonitrile-water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng. We derived the following stoichiometric equations: (see formula index).

Amylases↗
PubMed1978

Hydrolysis of aryl beta-maltotriosides by sweet potato beta-amylase and soybean beta-amylase.

Sweet potato beta-amylase [EC 3.2.1.2, alpha 1,4-D-glucan maltohydrolase]-catalyzed hydrolyses of aryl beta-maltotriosides with substituents, NO2-, Cl-, and Br- at the o-, m-, and p-positions in the phenyl ring were studied at pH 4.8 and 25 degrees C. The hydrolyses of a few of the maltotriosides by soybean beta-amylase [EC 3.2.1.2, alpha-1,4-D-glucan maltohydrolase] were also studied at pH 5.4 and 25 degrees C. It was found that the aryl beta-maltotriosides were preferentially hydrolyzed into maltose and aryl beta-D-glucosides by both beta-amylases. The Michaelis constant Km and the molecular activity ko were determined for the hydrolyses of these maltotriosides and compared with those of maltotriose and maltotetraose. Aryl beta-maltotriosides were more rapidly hydrolyzed than maltotriose by a factor of 30--80, and more slowly hydrolyzed than maltotetraose by a factor of 10--30, depending on the kinds of substituents. The rapid hydrolysis of aryl beta-maltotrioside as compared with maltotriose may be due to the interaction of an aryl group with the subsite of beta-amylase. This is in contrast with glucoamylase [EC 3.2.1.3, alpha-1,4-D-glucan glucohydrolase] of Rhizopus niveus-catalyzed hydrolysis of phenyl beta-maltoside, whose phenyl group does not interact so much with the subsite of the enzyme.

Amylases↗
PubMed1983

Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC.

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.

Amylases↗
PubMed1981

[The sequence of the alpha-amylase inhibitor Hoe-467 A (alpha-amylase inactivator Hoe-467 A) from Streptomyces tendae 4158].

An alpha-amylase inhibitor isolated from Streptomyces tendae, strain 4158 was re-purified by chromatography on CM- and DEAE-cellulose column. Two inhibitors could be characterized: alpha-amylase inhibitor Hoe-467 A (with aspartic acid as N-terminal residue), and alpha-amylase inhibitor Hoe-467 S (with serine as N-terminal residue). The primary structure was determined by automatic Edman-degradation procedures of the aziranized inhibitor and tryptic peptides, derived from digestions of the performic oxidized, aziranized and maleylated inhibitor, respectively. The alpha-amylase inhibitor Hoe-467 A consists of 74 residues and has a calculated molecular weight of 7958. It is composed of all common amino acids except methionine and phenylalanine. Digestion with pepsin was carried out to determine the disulfide bonds. Two fractions could be isolated, containing one cystine each giving information about the positions of the disulfide bridges. The possible clinical application of the inactivator (diabetes mellitus) is pointed out.

Amino Acid Sequence↗
PubMed1979

Amylase-producing ovarian neoplasm with pseudo-Meigs' syndrome and elevated pleural fluid amylase: case report and ultrastructure.

Elevated amylase activity was noted in the pleural effusion of a patient who was later found to have a stage I low-grade serous papillary ovarian neoplasm. The effusion resolved spontaneously after resection of the ovarian tumor, which contained large amounts of amylase activity. The ultrastructure of the tumor epithelium resembled that of normal salpinx. Secretory-type cells were present with apical, variably electron-dense secretory granules and cytoplasmic glycogen deposits. Amylase activity in the ovarian neoplasm probably resulted from the presence of functioning endosalpingeal-type epithelium in the tumor. In cases of effusion with unexplained amylase elevations, the possibility of serous ovarian neoplasia should be considered, even in the absence of demonstrable extra-ovarian dissemination.

Amylases↗
PubMed1979

The amylase-producing microflora of semi-preserved canned sausages: identification of the bacteria and characterization of their amylases.

Thirteen strains of amylase-producing bacteria were isolated from semi-preserved canned sausages and their ingredients. All belonged to the genus Bacillus, and could be separated into 4 different groups. Two groups were different strain of B. subtilis, one was B. amyloliquefaciens and the last was B. macerans. The identification of the different bacteria species was supported by disc gel electrophoresis of the supernatant culture fluid, after growth. The amylases were characterized with regard to temperature optimum, pH optimum and thermostability. Although some of the amylases appear to be quite thermostable, the only explanation for starch degradation in semi-preserved foods seems to be the amylase production from outgrowing spores which survived the heat treatment.

Amylases↗
PubMed1995

Location of the alpha-amylase gene in rumen Streptococcus bovis strains distinguished by unstable amylase activity.

Genetic stability of amylase activity after serial subcultivation experiments with amylolytic ruminal Streptococcus bovis strains was investigated. Two strains Amy+ and Amy- were obtained. Loss of amylase activity connected with the loss of plasmid DNA was not found in these strains. The presence of the gene responsible for the amylase activity in the chromosome of these strains was revealed by hybridization of the alpha-amylase gene on pJK108 against chromosomal DNA of S. bovis and Bacillus subtilis after a complete restriction with EcoRI.

Animals↗
PubMed1997

Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases.

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.

Amino Acid Sequence↗
PubMed1987

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene.

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.

Amino Acid Sequence↗
PubMed1988

Expression of the human amylase genes: recent origin of a salivary amylase promoter from an actin pseudogene.

The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. We have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5' regions of the five human amylase genes contain a processed gamma-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3' untranslated region of gamma-actin. In addition, insertion of an endogenous retrovirus has interrupted the gamma-actin pseudogene in four of the five amylase genes.

Actins↗
PubMed1990

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P.

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.

Binding Sites↗
PubMed1996

Interaction of allergens from house-dust mite and from cereal flours: Dermatophagoides pteronyssinus alpha-amylase (Der p 4) and wheat and rye alpha-amylase inhibitors.

Major wheat and rye allergens associated with baker's asthma have been identified as specific inhibitors of Dermatophagoides pteronyssinus alpha-amylase (Der p 4). the most active inhibitors were the wheat tetrameric one and the rye dimeric RDAI-1. these data suggest that interaction between mite and cereal allergens (alpha-amylase/inhibitor complexes) might occur in house-dust-mite-infected flours. In contrast to the strong inhibition of the d. pteronyssinus enzyme by the wheat and rye inhibitors, the amylases from D. farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae were much less affected. Moreover, the D. pteronyssinus amylase showed higher pH optimum (6.5 versus 5.0) and activity level (2-6 times/mg of protein in whole-mite extracts) than those from the three other mite species.

Allergens↗
PubMed1995

Characterization of a DNA fragment carrying the raw starch-digesting alpha-amylase and salt-dependent alpha-amylase genes from Bacillus circulans F-2.

A 5.4 kb HindIII DNA fragment carrying the gene encoding raw starch-digesting alpha-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli [Kim et al. (1990) Biochim. Biophys. Acta 1048, 2233-2238]. Interestingly, when the cell extract of E. coli harboring a plasmid carrying this fragment was incubated with 1 M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl. Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent alpha-amylase (SDA). Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl. SDA activity was fully detected at above 1 M NaCl. Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment.

Bacillus↗