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PubMed1986

Cholecystokinin receptors: relationships among phosphoinositide breakdown, amylase release and receptor affinity in pancreas.

The gut hormone cholecystokinin (CCK) octapeptide stimulates the release of amylase from exocrine pancreas, a process believed to be the result of the breakdown of phosphatidylinositol lipids. To examine further the relationship between phosphoinositide (PI) breakdown and amylase release, we have investigated the effect of N-terminally protected CCK C-terminal fragments in these systems using guinea-pig pancreatic lobules. There was a good correlation between the ability of these fragments to elicit amylase release and their potency in enhancing PI breakdown. In general, the EC50 for amylase release is 10-fold lower than the EC50 for PI breakdown. In addition, a good correlation existed between amylase release and the affinity of CCK fragments for [125I]Bolton Hunter-CCK octapeptide binding sites in pancreatic membranes. We also discovered that N-carbobenzyloxy-CCK tetrapeptide was relatively inefficient in causing PI breakdown, but it caused a near maximal stimulation in amylase release. These findings might reflect an amplification mechanism between PI breakdown and amylase release or that N-carbobenzyloxy-CCK tetrapeptide-induced amylase release is independent of PI breakdown.

Amylases↗
PubMed1987

Clinical significance of amylase isoenzyme determination.

Total amylase activity in serum and urine is formed by pancreatic (P) and salivary (S) isoenzymes. The evaluation of isoamylases provides better information on enzyme changes during the disease than total activities alone. The resolution of pancreatic from extrapancreatic origin of hyperamylasemia may be clinically important. The experience obtained from the analysis of isoamylases in more than 1500 patients with different clinical diagnoses we compare with a contemporary knowledge of disturbances in amylase activities. We developed a method separating quantitatively both isoamylases on the mini-columns of ion-exchanger which we used in routine clinical investigation. In the first section we selected the findings on physiology and biochemistry of isoamylases. We described for the first time a significant decrease of P-isoamylase activity in serum during the intravenous infusions of hypertonic glucose, amino acids and during acute hypercalcaemia. We suggested that hypertonic glucose, amino acids and calcium may regulate directly or indirectly the amylase flux from acinar cells in the pancreas across basolateral membrane into blood. This endocrine secretion of amylase may be important in different clinical conditions in which changes of neurohumoral and/or hormonal regulation are developed. The isoamylase activities in patients with different diagnosis are analyzed in the clinical section. The results may be correctly evaluated only in connection with the pathogenesis of isoamylase changes. Disorders of the organs producing amylase (i.e. pancreas or salivary glands) may induce changes of isoamylases depending on their functional status. A progressive loss of amylase producing cells may be accompanied by a decrease of enzyme activity in serum as was described in chronic pancreatitis with exocrine insufficiency. However, the amylase activity in serum is significantly influenced by clearance mechanisms, too. Disorders of the liver or kidneys are accompanied predominantly with hyperamylasemia caused by the disturbed clearance mechanisms. The amylase activity in serum is a consequence of the result between input and output of the enzyme within the blood stream. Some humoral and hormonal regulations are able to modulate both processes in vivo. We suppose that pathogenetic standpoint has the main role for correct interpretation of isoamylase activities. The pathogenesis of hyperamylasemia is therefore discussed in single chapters. In conclusion, the isoamylase activities in serum and urine are influenced beside genetic background by many factors in health and disease which may be respected during the evaluation of the results.

Amylases↗
PubMedDate not supplied

Low molecular organic inactivators in differentiating organ origin of alpha-amylase in humans. Part I.

Alpha-amylases derived from pancreatic juice, saliva and granulocytes show different sensitivity to the low molecular organic inactivators. Pancreatic alpha-amylase is more susceptible to inhibitory activity of d-glyceraldehyde and d-mannitol and glycerol in comparison to alpha-amylase from saliva and granulocytes. Iodoacetate and urea exhibited inhibitory activity against granulocytic alpha-amylase and only a minute inactivation of salivary and pancreatic alpha-amylases was found. The most effective inhibition against all tested alpha-amylases was noted with dihydroxyacetone. We should stress, no evident differences were noted in the degree of sensitivity to this inactivator of the tested enzymes. The presumable mechanism of the inactivation of alpha-amylases with low molecular organic inactivators is briefly discussed. The procedure for the differentiation of organ origin of alpha-amylases is suggested.

Dihydroxyacetone↗
PubMedDate not supplied

[Dependence of pancreatic amylase secretion on the amylolytic activity of the duodenal contents].

Pancreatic amylase secretion stimulated by intraduodenal administration of acidified (up to pH 2.0) hydrolysine was evaluated in acute experiments on dogs. Bicarbonates, protein, amylase, lipase, and proteinase were assayed in the juice. Renal excretion of amylase and amylolytic activity of blood plasma were determined during the experiment. Intraduodenal administration of pancreatic amylase decreased amylase secretion by the pancreas and did not influence the secretion of bicarbonates and other enzymes. Administration into the duodenum of amylase with its inhibitor eliminated the effect of selective inhibition of pancreatic amylase secretion. Amylolytic activity of the blood was unchanged, while the renal amylase excretion increased.

Animals↗
PubMed1982

Determinations of amylase isoenzymes in serum by use of a selective inhibitor.

A simple, rapid screening procedure for determining the relative amounts of pancreatic (P)- and salivary (S)-type amylase in serum is presented, involving incorporation of a selective inhibitor (from wheat-germ) into commercially available BMD Single-Vial Amylase and Beckman Enzymatic Amylase-DS procedures for manual and automated isoamylase measurements. Optimal concentrations of inhibitor inhibit the S-type amylase fraction by 87-88%. In contrast, the pancreatic fraction is inhibited by only 19% in either the manual or automated methods. The degree of inhibition is constant for amylase activities up to at least 520 U/L. Use of the ratio (P-amylase/total amylase activity) X 100 helps differentiate between hyperamylasemia caused by S-type or P-type amylase. In preliminary studies, patients with pancreatitis showed a ratio greater than 70%.

Amylases↗
PubMed1982

Improved DEAE-Sephadex column chromatography in measuring amylase in serous ovarian neoplasms, and results for 13 cases.

We describe a column-chromatographic method for measuring amylase activity in cyst fluids of serous ovarian tumors. Using this technique, we confirm and extend the previous report of an "acidic" amylase in serous ovarian tumor cyst fluids. This form of the enzyme was eluted from DEAE-Sephadex mini-columns with a high-salt buffer. It accounted for 45 to 100% of the amylase in 13 cyst fluids. This "high-salt" amylase was also present in the tumor tissues. The acidic nature of the amylase does not appear to be due to sialic acid residues, because the chromatographic behavior of the amylase is unaffected by treatment with neuraminidase. We conclude that the "acidic" amylase reported previously in two serous ovarian tumors is a constant feature of these tumors and that it is distinct from the sialylated tumor amylase described by others.

Amylases↗
PubMed1984

Expression and regulation of alpha-amylase gene family in barley aleurones.

Several alpha-amylase clones were identified by screening a group of 345 cDNA clones derived from poly(A)+RNA from gibberellic acid (GA)-treated barley aleurone layer cells using an alpha-amylase cDNA clone that we had characterized previously as the hybridization probe. The alpha-amylase clones showed differences in the distribution of restriction enzyme sites, in the extent of cross hybridization, and in nucleotide sequence. There are at least three types of alpha-amylase clones in our collection, and the alpha-amylase cDNA clone reported by Rogers and Milliman [J. Biol. Chem. (1983) 258: 8169-8174] is a fourth type. The accumulation of alpha-amylase mRNAs in aleurones as a function of time after GA addition and of hormone concentration in the incubation medium was studied using different alpha-amylase cDNA clone probes. These studies indicate a differential control of the expression of the alpha-amylase genes by GA.

Base Sequence↗
PubMed1994

Catalytic subunit of protein kinase A induces amylase release from streptolysin O-permeabilized parotid acini.

Amylase release from parotid acinar cells is a typical model of cAMP-mediated exocytosis. To obtain unequivocal data concerning the role of cAMP-dependent protein kinase (PKA) in amylase exocytosis, we undertook the direct introduction of the PKA catalytic subunit into the parotid acini by permeabilization with streptolysin O (SLO). In the presence of 100 hemolytic units/ml SLO, cAMP increased amylase release in a time- and dose-dependent manner. PKI-(5-24)-peptide, a specific PKA inhibitor, markedly inhibited amylase release, but the extent of inhibition was approximately 50%. On the other hand, the PKA catalytic subunit highly purified from bovine hearts significantly induced amylase release. The release was strictly dependent on the presence of SLO and the catalytic activity of PKA added. The catalytic subunit dose dependently induced amylase release, but the heat-inactivated subunit had no stimulatory effect. PKI-(5-24)-peptide completely blocked amylase release evoked by the subunit. These results clearly demonstrate that the catalytic subunit of PKA regulates cAMP-mediated amylase release through phosphorylation of unidentified protein(s) directly or indirectly involved in the process of exocytosis.

Amylases↗
PubMed1995

Fluctuations in serum amylase in patients with macroamylasemia.

OBJECTIVE: To report wide fluctuation of serum amylase in patients with macroamylasemia. It has generally been considered to remain constant. METHODS: Over the past 16 y, 18 patients have been diagnosed with macroamylasemia in our GI department. Of these, four patients were followed up with serial serum amylase determinations for a period of less than 1-4 y. Serum amylase was measured by the "Phadebas amylase test." Serum macroamylase was measured by "PEG precipitation technique." RESULTS: There was a wide fluctuation of serum amylase in three out of four patients. In the fourth patient, more persistent hyperamylasemia was noted during the shorter observation period. CONCLUSION: Marked fluctuation in serum amylase, ranging from 115 to 1160% in this study, may occur in patients with macroamylasemia. The reasons for these fluctuations are not clear but may be due to association-dissociation of amylase with serum proteins at variable time intervals. This fluctuation, especially when the amylase becomes normal (as in cases 1 and 3), may lead to confusion in differentiating macroamylasemia from other causes of hyperamylasemia.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Hybrid Bacillus amyloliquefaciens X Bacillus licheniformis alpha-amylases. Construction, properties and sequence determinants.

A series of 33 single and mosaic hybrid alpha-amylases was constructed from the genes amyBA or amyLI, encoding the alpha-amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus licheniformis (AmyLI). The hybrid proteins, consisting of the entire alpha-amylase sequence with a variable portion of AmyBA or AmyLI origin, were characterized in order to find enzymes with new properties (thermostability, temperature and pH optima, and substrate specificity), and to localize the amino acid sequence regions responsible for the changes. The thermostability of the AmyBA/AmyLI (AL-type) hybrid proteins correlated with the position and the length of the hybrid sequence. The hybrid enzymes fell into six groups retaining, in comparison to AmyBA, a certain value of the extra-thermostability of AmyLI or becoming more thermolabile than AmyBA. Four regions are proposed to contain thermostability determinants (TSDs). They map between amino acid residues 34-76, 112-142, 174-179 and 263-276 of the respective hybrid enzymes, indicating the dominance of the N-terminal half of AmyLI for these hybrid enzymes' resistance against irreversible inactivation. Two (TSD3 and TSD4) coincide with regions I and II that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chem. 264, 18,933-18,938]. The temperature dependence of activity of the AL-type hybrid alpha-amylases was compared at pH 6.4 and pH 7.6 and the hybrid enzymes of one thermostability group were found to have similar temperature responses. A hybrid region between residues 34-76 is demonstrated to correlate with the alpha-amylases' substrate specificity, i.e. either hydrolysis or accumulation of maltohexaose. This region was therefore named the G6G5 region. The exchange of internal sequences between residues 17-201 of AmyBA by the AmyLI counterpart in ALA-type mosaic hybrid alpha-amylases, with one exception (ALA99-429), unexpectedly destabilized the respective ALA-type hybrids. Two of these hybrid alpha-amylases (ALA17-151 and ALA76-151) were less thermostable than AmyBA, while others (ALA112-151, ALA112-201) were enzymically inactive. These data support specific roles of the predicted A1-B domain portion between residues 17-201 of those Bacillus alpha-amylases probably for correct folding and enzymic activity.

Amino Acid Sequence↗
PubMed1996

Thermostable alpha-amylase production using Bacillus licheniformis NRRL B14368.

Maximum amount of extracellular alpha-amylase of B. licheniformis NRRL B14368 was obtained at the stationary phase. Highest yield of alpha-amylase was achieved with high level of crude protein and low carbohydrate level. There was a catabolite repression in the organism. Protease was produced concurrently with alpha-amylase. It was also observed that soyabean acts as an inhibitor of the protease. Optimum pH and temperature of alpha-amylase were 5-7 and 76 degrees C respectively. It was also observed that alpha-amylase production was a non-growth associated product. Maltose was an excellent inducer for alpha-amylase production. Ca2+ (0.01 M) increased the thermostability of the enzyme. Alpha-amylase purification studies were carried out by using isopropanol, acetone, ammonium sulphate solution, ion exchange chromatography. Acetone was found most suitable for the separation of alpha-amylase. Protein recovery and relative enzyme activity (as compared to that of the maximum activity of the crude enzyme) were 30.77% and 3.03 respectively.

Bacillus↗
PubMed1997

beta-Amylase production by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [correction of polymaxa] strains.

The production of extracellular beta-amylase by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [corrected] strains was investigated, and the maximal yields of the enzyme were 3.6; 9.3 and 20.4 U/mL of the culture fluid, respectively (U, 1 mumol of maltose equivalent per min at 30 degrees C). Several cultivation media were used for beta-amylase production. Bacillus cereus and some strains of Bacillus megaterium gave good yields of beta-amylase only in medium with the addition of nutrient broth. However, beta-amylase produced during growth in protein rich medium (nutrient broth) was highly unstable, probably due to inactivation by proteolytic enzymes co-existing in the culture fluid. Bacillus polymyxa [corrected] strains can produce good yields of beta-amylase on a semi-synthetic medium consisting of inorganic salts, potato starch and inexpensive soybean extract instead of costly peptone and meat extract. The most potential beta-amylase producer was the strain Bacillus polymyxa [corrected] NCIB 8524. The tested Bacillus megaterium and Bacillus polymyxa [corrected] strains were apparently differentiated by temperature cultivation (30 and 37 degrees C) suitable for beta-amylase amylase yield.

Bacillus↗
PubMed1984

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2.

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.

Amylases↗
PubMed1999

The concept of the alpha-amylase family: structural similarity and common catalytic mechanism.

This review reconsiders the concept of the alpha-amylase family in the light of the recent wealth of information on the structures, the catalytic mechanisms, and the classification of amylases. We proposed a general concept for an enzyme family, the alpha-amylase family including most of the amylases and related enzymes in 1992, based on the structural similarity and the common catalytic mechanisms. The study on neopullulanase was the key to open the door for the formulation of the concept. We discovered a new enzyme, neopullulanase, and proved that the enzyme catalyzes both hydrolysis and transglycosylation at alpha-1,4- and alpha-1,6-glucosidic linkages by one active center. Results from a series of experiments using neopullulanase indicated that the four reactions mentioned above could be catalyzed in the same mechanism. Progress in X-ray crystallographic analysis has allowed researchers to observe the structural similarities among alpha-amylases, cyclodextrin glucanotransferases, and an isoamylase. The primary structural analyses and the secondary structural predictions also suggest a close relationship among enzymes with three-dimensional structures which catalyze one of the four reactions. They possess a catalytic (beta/alpha)8-barrel as observed in the crystal structure of alpha-amylases, cyclodextrin glucanotransferases, and an isoamylase. Two crucial points, the common catalytic mechanisms and the structural similarities among the enzymes which catalyze the four reactions, led us to propose the concept of the alpha-amylase family. We would like to point out the significance and problems of the sequence-based classification of glycosyl hydrolases. The possible catalytic mechanism of the alpha-amylase family enzyme is also described for the rational design of tailor-made artificial enzymes.

Journal Article↗
PubMed1975

Genetic control of endosperm amylase activity and gibberellic Acid responses in standard-height and short-statured wheats.

In contrast to standard-height wheat genotypes, short-statured wheats having major genes for dwarfness do not show increased seedling growth after treatment with gibberellic acid. Endogenous gibberellic acid induces synthesis of amylase in the endosperm of germinating seeds, but the amount of amylase synthesized is greatly increased by exogenous gibberellic acid treatment in standard-height and in short-statured wheats that have dwarfing genes from the variety "Norin 10." "D6899," which has the "Tom Thumb" gene for height reduction, had about one-fourth of the amylase activity of standard-height and Norin 10-derived, short-statured wheats. This genotype showed little or no increased amylase activity after gibberellic acid treatment. Genetic analyses showed that the amount of amylase synthesized was controlled by a single gene and was dependent on the number of copies of the structural gene present in the endosperm. Dwarfism in wheat may be related to a blockage in gibberellic acid utilization because other workers have found that the amount of endogenous amylase synthesized in Norin 10-derived, short-statured wheats is not growth-limiting, but it is not known if low amylase synthesis is related to dwarfism in the Tom Thumb derivative. No recombinants were recovered in a small population, suggesting that the Tom Thumb gene may pleiotropically affect plant height and the lack of response to gibberellic acid in amylase synthesis and seedling growth.

Journal Article↗
PubMed1993

Aleurones from a Barley with Low [alpha]-Amylase Activity Become Highly Responsive to Gibberellin When Detached from the Starchy Endosperm.

The physiological and molecular bases for contrasting [alpha]-amylase phenotypes were examined in germinating seeds of two barley (Hordeum vulgare L.) cultivars, Morex and Steptoe. Morex is a high-quality malting barley that develops high [alpha]-amylase activity soon after germination. Steptoe is a feed barley that develops only low [alpha]-amylase activity levels during this period. The expression of all high- and low-isoelectric point (pl) [alpha]-amylase isozymes is reduced in Steptoe. The amount of [alpha]-amylase mRNA per gram of seedling tissue is correspondingly lower in Steptoe. Southern blot analysis revealed that the cultivars have the same copy number and organization for most high- and low-pl genes. Steptoe seedlings or embryoless half-seeds produce little [alpha]-amylase in response to exogenous applications of gibberellic acid (GA3) compared with Morex. However, when isolated aleurones of both cultivars are treated with GA3, they produce similar amounts of high- and low-pl [alpha]-amylase RNAs. This suggests that a factor in the starchy endosperm is responsible for lowered [alpha]-amylase response in Steptoe. The factor is probably not abscisic acid (ABA), since the two cultivars have similar concentrations of ABA during germination.

Journal Article↗
PubMed1970

alpha-Amylase Isozymes in Gibberellic Acid-treated Barley Half-seeds.

The presence of multiple forms of alpha-amylase in gibberellic acid-treated embryoless barley half-seeds was demonstrated by separation on diethylaminoethyl-Sephadex and isoelectric focusing polyacrylamide gel disc electrophoresis. Two major alpha-amylase fractions (A and B), each consisting of two to three isozyme components, were purified. alpha-Amylase fractions A and B were distinguishable in their reaction patterns. The optimal pH of fraction A alpha-amylase was found to reside in the acidic side (pH 5.0), as was determined by analyzing the reducing sugars formed as well as the paper chromatographic detection of reaction products. At neutral pH, 6.9, fraction A exhibited weak amylolytic activity in forming maltose. The alpha-amylase activity in fraction A was markedly stimulated by heat treatment (70 C/15 minutes). Fraction B, constituting a major part of amylases in the endosperm extract, was also found to be composed of alpha-amylase, as evidenced by the loss of enzyme activity upon allowing fractions A and B to stand at pH 3.3 for a prolonged period. The possible physiological function of the two different types of alpha-amylase in the carbohydrate breakdown of barley seeds is discussed.

Journal Article↗
PubMed1982

In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS.

Wheat (Triticum aestivum) RNA was used to program synthesis of the alpha-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as alpha-amylase by immunoprecipitation with rabbit anti-alpha-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic alpha-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of alpha-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of alpha-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the alpha-amylase protein to a form which co-migrated with authentic alpha-amylase purified from malted wheat and with the protein synthesized in oocytes.

Journal Article↗