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PubMed1991

[A thermostable alpha-amylase from Thermoactinomyces vulgaris: purification and characterization].

Alpha-amylase from Thermoactinomyces vulgaris (strain 94-2A) was purified by cellulose chromatography and gel-chromatography on Sephadex G-75 and subsequently characterised. The enzyme shows a single band in the polyacrylamide gel electrophoresis (PAGE). The isoelectric point was determined to be pH 5.4, and the molecular mass was estimated as 53,000 Dalton by PAGE. The amino acid composition was determined; it shows characteristics of other microbial alpha-amylases. A comparison of the N-terminal sequence with that of other alpha-amylases shows a homology of 66.6% to Taka-amylase. The pH-optimum for the alpha-amylase activity is 4.8 to 6.0 and the temperature optimum 62.5 degrees C. The heat inactivation was investigated under different conditions (temperature, time, Ca2+, EDTA).

Amino Acid Sequence↗
PubMed1976

Possible mechanisms of normal amylase activity in hyperlipemic pancreatitis.

Lipemic serum from three patients with acute pancreatitis and type IV hyperlipemia was fractionated into very-low-density lipoproteins and clear serum. Amylase activity (determined by the Phadebas method) in the component fractions did not exceed that in the original lipemic serum. Addition of these fractions or VLDL and chylomicrons from asymptomatic patients with hyperlipemia to nonlipemic serum from patients with "routine acute pancreatitis" did not inhibit amylase activity or alter the electrophoretic mobility of amylase isoenzymes. Therefore the normal amylase activity often observed in hyperlipemic pancreatitis does not result from an inhibition of amylase activity by serum lipoproteins.

Acute Disease↗
PubMed1990

Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133.

A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.

Amino Acid Sequence↗
PubMed1975

Purification and properties of phaseolamin, an inhibitor of alpha-amylase, from the kidney bean, Phaseolus vulgaris.

Kidney beans, Phaseolus vulgaris, contain a proteinaceous inhibitor of alpha-amylase, which we have named phaseolamin. The inhibitor has been purified to homogeneity by conventional protein fractionation methods involving heat treatment, dialysis, and chromatography on DEAE-cellulose, Sephadex G-100, and CM-cellulose. Phaseolamin is specific for animal alpha-amylases, having no activity towards the corresponding plant, bacterial, and fungal enzymes, or any other hydrolytic enzyme tested. Optimal inhibitory activity is expressed during preincubation of enzyme and inhibitor at pH 5.5 and 37 degrees. Substrate prevents inhibition. Measurement of the stoichiometry on inhibition showed that a 1:1 complex of alpha-amylase and inhibitor is formed. Complex formation was demonstrated by chromatography on Sephadex G-100. The phaseolamin-amylase complex is dissociated at low pH values, apparently as a result of destruction of the enzyme; the complex cannot be dissociated by other conditions unfavorable for inhibition (low temperature or high pH). Phaseolamin inhibits hog pancreatic alpha-amylase in a noncompetitive manner.

Amylases↗
PubMed1986

Three methods compared for determination of pancreatic and salivary amylase activity in serum of cystic fibrosis patients.

We evaluated three methods for serum amylase (EC 3.2.1.1) isoenzymes to determine whether they are interchangeable and to test their ability to discriminate between cystic fibrosis patients with and without pancreatic insufficiency. One method involved salivary amylase inhibitor (O), and two were polyacrylamide gel electrophoresis separations differing in method of detection--either direct zymogram (G) or gel slicing followed by activity estimates per slice (W). Results for percentage pancreatic amylase differed significantly. Reproducibility for percentage pancreatic amylase was high, moderate, and low (r = 0.95, 0.53, and 0.02) for methods G, O, and W, respectively; moderate (r = 0.60) among the three methods; and moderate between pairs. Therefore, this result for a subject must be considered relative to the method used in its determination. The clinical diagnosis of pancreatic insufficiency was verified by 77.8%, 83.3%, and 94.4% correct classification rates for methods O, W, and G, respectively. Evidently, method G is the most efficient and may be the method of choice for measuring serum amylase isoenzymes in cystic fibrosis.

Adolescent↗
PubMed1986

Effect of perioperative somatostatin administration of sphincteroplasty-induced increase of amylase.

Twenty patients undergoing sphincteroplasty for cholelithiasis were randomly divided into two groups of 10. The former (T) were treated with a 4-h somatostatin intravenous drip (250 micrograms/h), started at the beginning of operation, while the latter (C) made up the control group. Serum and urine amylase, amylase creatinine clearance ratio, and liver function tests were assessed for 2 days before surgery, after the operation and for a period of 5 postoperative days. Homogeneity between the two series was verified in experimental conditions. Statistical differences occurred postoperatively in amylase creatinine clearance ratio, which proved higher in C group, and gamma-GT, which was higher in T group. Short-term somatostatin administration proved effective in reducing the postoperative amylase creatinine clearance ratio, although more evident results are reported after long-term administration. Cholestasis or any serious impairment in liver function did not occur, suggesting the suitability of somatostatin use even in patients with jaundice. Since a relationship between postoperative amylase levels and risk of pancreatitis has not yet been proved, the value of somatostatin in the prevention of postoperative pancreatitis after sphincteroplasty needs to be further verified.

Aged↗
PubMed1986

Determination of amylase activity in serum by using a wheat germ inhibitor with the Du Pont aca.

A rapid procedure for determining salivary- and pancreatic-type amylase (EC 3.2.1.1) in serum by incorporating a wheat germ inhibitor (from Triticum aestivum) was developed for the Du Pont aca IV analyzer. Under optimal assay conditions, activities of salivary and pancreatic amylase were inhibited by 93% and 19%, respectively. The 95% central reference interval for the percentage of inhibition of serum amylase was 38-84%. Patients with acute pancreatitis showed less than 26% inhibition of amylase after addition of the wheat germ extract, reflecting the prevalence of pancreatic-type amylase in this disorder.

Acute Disease↗
PubMed1986

[Selectivity of inhibition of enzyme secretion by the pancreas after introduction of amylase into the duodenum].

Intraduodenal administration of both pancreatic and vegetative amylase induces inhibition of pancreatic secretion of the amylase. The secretion activated with intraduodenal administration of oxidized hydrolysine or i.v. administration of secretin, is also inhibited. High doses of intraduodenal amylase inhibit secretion of the juice's other components too. I.v. amylase inhibit secretion of bicarbonates and hydrolases in the pancreas. The selective inhibiting effect of amylase is sharply reduced with starch solution. The duodenum seems to be able to inhibit selectively the pancreatic enzymes secretion, this mechanism participating in urgent adaptations of the pancreatic juice enzyme range to properties of duodenal chyme.

Amylases↗
PubMed1986

[Determination of amylase creatinine clearance in children and adolescents].

UNLABELLED: 24 children whose ages ranged from 10 days to 14 years (x 5.5. ys.), 16 males and 8 females, hospitalized in our Service on account of extra-digestive were studied. Creatinine and amylase determination were performed on serum and 24 hs. urine specimens; lipase activity was measured only in serum. Amylase-creatinine clearance ratio varied between 1.3 and 5.8% (x 3.1 +/- 1.3). Serum amylase ranged from 36 to 460 U/l (x 123 U/l). Five patients had hyperamylasemia. 2 were urlian parotiditis, 2 were under clinical surveillance after surgery and the last one was a septic meningitis. All of them showed amylase-creatinine ratio values up to 2.5%. Serum lipase ranged from 6 to 197 U/l (x 79 U/l), falling into the normal interval. No abnormal clearance was found. CONCLUSIONS: hyperamylasemia is not specific for pancreatic disturbances in children; the serum lipase and the amylase-creatinine clearance ratio seem to be useful tools to rule out pancreatic involvement in hyperamylasemic patients.

Adolescent↗
PubMed1987

Expression of vasoactive intestinal polypeptide and amylase in a human parotid gland adenocarcinoma cell line in culture.

A neoplastic epithelial cell line initially established in culture from a human parotid gland adenocarcinoma grown in athymic nude mice with a BALB/c genetic background, which has an ultrastructure similar to that of the intercalated duct cell of the salivary glands, was examined for the expression of amylase and vasoactive intestinal polypeptide (VIP). The cultured cells were found by the peroxidase-antiperoxidase (PAP) method to express amylase and ultrastructurally to have secretory granules showing positive immunoreaction with anti-amylase serum. In addition, the cells were found to secrete amylase into the culture medium. The expression of VIP in the cultured cells was observed by the PAP method, immunofluorescent staining method, or immunoelectron microscopy. Moreover, the presence of the polypeptide reactive to antibodies directed against VIP in the cultured cells was confirmed by immunoblotting and radioimmunoassay. These findings indicate that the cells proliferating in culture express both amylase and VIP.

Adenocarcinoma↗
PubMed1988

Amylase in urine as measured by a single-step chromolytic method.

We studied a new single-step direct chromolytic method (Behring D.A.T.) for measuring urinary amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) activity, comparing results with those by a similar, but two-step, procedure that requires an auxiliary (coupling) enzyme. The two methods gave virtually identical relative responses to purified human pancreatic and salivary amylases. Assay of four quality-control materials to evaluate the total (day-to-day) precision of the new method yielded CVs of 4 to 7%, similar to those of the comparison method for each of the four quality-control samples. Amylase activity was measured by both methods in 110 random (i.e., untimed) urine specimens. Linear regression analysis provided a slope and y-intercept of 0.947 and 4 U/L (x = comparison method, y = direct method), respectively, and a standard error of the estimate of 25 U/L for specimens in which the amylase activities ranged from 11 to 1465 U/L (mean = 358 U/L) by the comparison method. The mean rate of amylase excretion in 2-h timed urine specimens from 95 healthy volunteers, as measured by the new method, was 7.18 (SD = 3.18) U/h, and the nonparametric (95% confidence interval) reference interval was 1.6 to 15.2 U/h. We consider the new method a promising alternative to other kinetic assays that require the use of auxiliary enzymes.

Amylases↗
PubMed1989

[Amylase-producing lung tumors].

Raised serum amylase associated with pulmonary tumours is a rare phenomenon. On the basis of two cases, the literature is reviewed and the case histories are compared. The majority of tumours associated with raised serum amylase are adenocarcinomata and, in all of the cases in which determinations of isoenzymes were undertaken, the amylase-isoenzyme was of salivary gland type. In one of the authors' cases, immunhistochemical investigation revealed that the tumour cells contained amylase. In cases of hyperamylasaemia of unknown origin with increased amylase of salivary gland type, adenocarcinoma in the lungs should be considered as a possible diagnosis.

Adenocarcinoma↗
PubMedDate not supplied

[Two-codon mutagenesis of alpha-amylase gene of Bacillus amyloliquefaciens].

The oligonucleotide encoding Bam HI recognition site having the structure pCGGGATC had been inserted into the recognition sites MspI of the B. amyloliquefaciens alpha-amylase gene, which was cloned in pTG29B plasmid. The alpha-amylase gene had no BamHI sites before mutagenesis. The set of pNSBamHI plasmids with BamHI site at four different positions was obtained. It was shown that all the mutant alpha-amylases possess different specific activities. One of the mutant proteins possesses reduced thermostability. The mutant alpha-amylases can be used for further experiments on protein-engineering of liquefying-type alpha-amylases.

Bacillus↗
PubMed1989

[Separation of alpha-amylase isoenzymes using cellulose acetate electrophoresis].

alpha-Amylase isozymes were separated by electrophoresis in cellulose acetate to detect the isoforms of the chromogenic substrate manufactured by Lachema, Czechoslovakia. In a group of 20 normal subjects aged 25 to 45, alpha-amylase pancreatic isozyme activity prevailed. Patients with acute myocardial infarction developed, during 36 hours after the onset of the anginal attack, a reduction in the ratio of pancreatic amylase/salivary amylase activities in both increased and normal total alpha-amylase activities. The suggested modified technique of electrophoretic separation of alpha-amylase isozymes may become an effective method for differential diagnosis. Use of this method will help locate the source of hyperamylasemia in various diseases and will thus specify the diagnosis, ruling out the useless therapy for pancreatitis.

Adult↗
PubMedDate not supplied

[Enzymatic and conformational stability of polymeric complexes of alpha-amylase].

The enzymatic and conformational stability of Bacillus subtilis alpha-amylase and its polymeric complexes in acid media and subsequent renaturation in weakly alkaline media were investigated. The following parameters of alpha-amylase secondary structure were determined from circular dichroism spectra: helical units -25%, beta-structures -9%; beta-turns -13%; disordered conformations -53%. After complexation with polymethacrylic acid (PMAA) the alpha-amylase secondary structure did not change, and the tertiary structure underwent only small local changes. Complexation of alpha-amylase with linear and cross-linked PMAA led to an increase in both enzymatic and conformational stabilities in acid media. Purification of alpha-amylase using a biosorbent resulted in higher acid resistance of the free enzyme and of that in the complex with PMAA. Moreover, the degree of reversibility of the acid inactivation also increased.

Bacillus subtilis↗
PubMed1986

[Proteolytic susceptibility of alpha-amylase of the pancreas of swine deprived of its structural calcium].

Hog pancreas alpha-amylase (alpha-1-4-glucan-glucan hydrolase, E.C. 3.2.1.1) lost its structural calcium by action of EDTA at 20 degrees C. Enzymatic activity experimented a decrease whereas a big increase in proteolytic susceptibility to bovine pancreas trypsin (E.C. 3.4.4.4) was shown. Native alpha-amylase had an activity of 2,730 mg maltose/min X mg enzyme and a Km of 0.222% amylose, the activity of calcium depleted amylase being of 1,640 mg maltose/min X mg enzyme and Km 0.571% amylose. Simple methods for evaluating proteolytic susceptibility of alpha-amylase micro-amounts against trypsin action, and for the measurement of alpha-amylase activity in polyacrylamide rod gels were also described.

Animals↗
PubMed1987

A monoclonal antibody that specifically inhibits human salivary alpha-amylase.

Our monoclonal antibody 88E8 specifically binds to and inhibits human salivary alpha-amylase (EC 3.2.1.1) and cross reacts negligibly with the pancreatic isoenzyme, inhibiting it by less than 1%, as compared with about 90% for the salivary isoenzyme. The antibody binds the S1 and S2 types of salivary alpha-amylase, but no pancreatic alpha-amylase isoenzyme forms. A pancreatic alpha-amylase assay involving 88E8 is under development, with alpha-glucosidase as auxiliary enzyme and p-nitrophenyl-maltoheptaoside as substrate; we give preliminary data on this assay. The assay has to be done by substrate start, because the antibody interacts very slowly with the enzyme in the presence of substrate. Assay results for pancreatic alpha-amylase correlate well with those for isoamylase assayed with use of an inhibitor from wheat-germ.

Antibodies, Monoclonal↗
PubMed1985

Fluorescence depolarization assay for quantifying alpha-amylase in serum and urine.

We have developed a new method for quantifying alpha-amylase (EC 3.2.1.1) in serum and urine by fluorescence depolarization. Amylase in the sample catalyzes the hydrolysis of the substrate, a fluorescein-labeled amylose. This results in decreased fluorescence polarization, owing to the increased rate of rotation of the amylose fragment relative to the intact substrate. The TDx amylase assay is calibrated with six human-serum-based pancreatic amylase calibrators. Amylase activities are determined by interpolation from the calibration curve, which is stored in the TDx analyzer's memory. Results correlate well with those by the Du Pont aca assay and the Beckman "DRI-STAT" assay. Endogenous glucose does not interfere. CVs are less than 6%, and the reagents are stable in liquid form.

Fluorescence Polarization↗