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PubMed1979

The predominantly nonhydrolytic action of alpha amylases on alpha-maltosyl fluoride.

Crystalline alpha amylases from a number of sources utilized alpha-maltosyl fluoride as a glycosyl donor and acceptor at high rates (approximately 10 to approximately 1550 mumol/min/mg of protein, for 30 mM substrate). All enzymes catalyzed conversion of this compound into maltooligosaccharides in preference to causing its hydrolysis. Maltotetraosyl flouride and maltooligosaccharides of d.p. 3 to 6+ accounted for 75--93% (by weight) of early reaction-products. At a late stage, the yield of maltooligosaccharides was 2--5 times that of maltose, with chains as long as 12 D-glucosyl residues formed by one amylase (from Asp. oryzae), which utilized alpha-maltosyl fluoride as a donor and as an acceptor at extremely high rates. These results indicate that alpha amylases have a substantial capacity for binding two molecules of this small substrate in a distinctive way, with the C--F glycosylic bond of one and the free C-4 hydroxyl group of the other located in the region of the enzyme's catalytic groups, therby favoring glycosylation of the suitably positioned acceptor over solvent water. Hydrolysis is assumed to prevail when only a single substrate molecule or segment binds to alpha amylase with a (1 linked to 4)-alpha-D-glucosidic linkage of glycosylic C--F bond positioned at the catalytic center. The present demonstration that glycosyl-transfer reactions can be dominantly expressed by alpha amylases, given an appropriate substrate, illustrates the inadequacy of the usual characterization of these enzymes as hydrolases that produce overwhelming hydrolysis of all substrates.

Amylases↗

PubMed1979

Liposome-incorporated enzymes: studies on amylase.

Alpha-amylase (EC 3.2.1.1) from hog pancreas was incorporated into artificial phosphatidylcholine and phosphatidylserine liposomes. To kinetically follow the enzyme-catalyzed hydrolysis of amylose the liposome-bound amylase was incubated in a medium containing amylose-iodine substrate. The reaction was studied at different concentrations of amylose and at different ionic strengths. Activation of amylase incorporated into liposomes by chloride ions varies with the type of phospholipid utilized to prepare the liposomes. Whe amylase bound to negative charged liposomes was used, a sigmoidal relationship between the reaction velocity and substrate concentration was found. Incorporation into liposomes protects amylase from heat inactivation.

Amylases↗

PubMed1978

An automated method for determination of serum and urine alpha-amylase.

An automated method for the determination of alpha-amylase activities of serum and urine using the Phadebas Amylase Test is described. The procedure has been adapted for the System Olli 3000 analyser using amylase tablets whose weight is half of that of normal tablets. The results are calculated by the computer with the aid of a standard curve calculated theoretically. This automated procedure is fast, as many as 120 analyses per hour can be carried out. Intra-assay precision of 2.0 and 2.2% (CV) is obtained from serum samples containing 301 and 181 u/l of alpha-amylase, respectively (n = 20), and inter-assay precision is 4.4 and 3.3% with mean values of 337 and 196 u/l, respectively (n = 20). When the automated procedure is compared with the manual procedure on 85 sera with alpha-amylase activities below 1000 u/l, a good correlation r = 0.992, and a regression equation y = 1.01x-6 are found. In the case of serum and urine samples, which contain high enzyme activities, the automated method gives slightly higher results than the manual method.

Amylases↗

PubMed1982

Two distinct pancreatic amylase genes are active in YBR mice.

The genetic determinants of pancreatic amylase expression in YBR mice differ in two respects from those of other inbred strains. First, there are two nonallelic amylase isozymes present in YBR pancreas, while most mouse strains express a single pancreatic amylase protein. In addition, the in vivo rate of total pancreatic amylase synthesis is 50% of that in other strains. Both these traits are determined by genetic sites in the region of the Amy-2 locus on mouse chromosome 3. To determine the molecular basis for the presence of two isozymes in this strain, we have compared portions of their amino acid sequences. Two differences between isozymes A1 and B1 were identified among the 77 residues compared. This result demonstrates that two distinct amylase genes are expressed in YBR pancreas.

Amino Acid Sequence↗

PubMed1983

Population genetics of Drosophila amylase. IV. Selection in laboratory populations maintained on different carbohydrates.

Two polymorphic systems impinging on alpha-amylase in Drosophila pseudoobscura have been studied in laboratory populations maintained on medium in which the only carbohydrate source was starch (the substrate of amylase) and replicas maintained on medium in which the only carbohydrate source was maltose (the product of amylase). The two polymorphic systems were alleles at the structural gene (Amy) coding for the enzyme (allozymes) and variation in the tissue-specific expression along the adult midgut controlled by several genes. In the seven populations on maltose medium little consistent change was noted in either system. In the seven populations on starch medium, both polymorphisms exhibited selective changes. A midgut pattern of very limited expression of amylase rose in frequency in all starch populations, as did the frequency of the "fast" (1.00) Amy allele. The overall specific amylase activity did not differ between starch-adapted and maltose-adapted flies.--The results, along with previous studies, indicate that when a gene-enzyme system is specifically stressed in laboratory populations, allozymes often exhibit selective differences. Such results make the selectionist hypothesis at least tenable. Furthermore, the fact that both types of polymorphisms responded to selection indicates the role of structural gene vs. gene regulation changes in adaptive evolution is not an either/or question but one of relative roles and interactions.

Alleles↗

PubMed1983

Interaction of native and SH-modified beta-amylase of soybean with cyclohexadextrin and maltose.

Cyclohexadextrin and maltose bound to soybean beta-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra in the ultraviolet region. The difference spectrum produced by cyclohexadextrin, a competitive inhibitor, had peaks at 285, 292, and 299 nm, while that by maltose, a reaction product, had peaks at 285 and 292 nm and a small trough at around 300 nm. By using the peaks at 292 and 299 nm, the dissociation constants of enzyme-cyclohexadextrin and enzyme-maltose complexes were calculated to be 0.35 mM and 8.1 mM, respectively. The effects of modification of SH groups of beta-amylase on the interaction of the enzyme with these sugars were examined by using beta-amylase carboxymethylated at the SH1 site and the enzyme modified at SH1 and SH2 sites with iodoacetamide or with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The dissociation constants of the enzyme-cyclohexadextrin and enzyme-maltose complexes were not changed by the modification of these SH groups, but the modification of SH2, the so-called essential SH group of soybean beta-amylase, strongly affected the difference spectra produced by maltose. The spectrophotometric titration of beta-amylase by cyclohexadextrin in the presence of maltose showed that cyclohexadextrin and maltose bind to the enzyme competitively, regardless of the modification of SH2. These results indicated that SH2 is located near the binding site of cyclohexadextrin and maltose, but is not involved in the binding of these sugars.

Amylases↗

PubMed1981

Dissociation between stimulant-evoked acinar membrane resistance change and amylase secretion in the mouse parotid gland.

1. Isolated segments of mouse parotid gland were superfused with a physiological saline solution. Membrane potential and input resistance were measured with intracellular micro-electrodes. Amylase secretion was monitored using an automated fluorometric assay. The parotid gland was stimulated by exposure to isoprenaline (10(-6)M), ACh (10(-6) M), dibutyryl or monobutyryl cyclic AMP (10(-3)M). 2. ACh evoked a sharp decrease in input resistance (from about 3-6 M omega to 1-2 M omega ) accompanied by a modest (two-fold) increase in amylase output. Both effects were fully reversible. 3. Isoprenaline evoked a marked increase in amylase output (five to ten-fold) reaching maximum after 20-30 min of stimulation. Throughout the period of intensive secretion the input resistance remained at the control level. Short pulses of ACh stimulation dramatically reduced input resistance in this period. 4. Dibutyryl cyclic AMP and monobutyryl cyclic AMP caused marked increases in amylase output (five to ten-fold) peaking 40-50 min after start of the continuous stimulation. This increase in amylase secretion was not accompanied by any change in input resistance. Short pulses of ACh stimulation sharply and reversibly reduced input resistance both in control and test periods. 5. It is concluded that the ACh-evoked reduction in input resistance is not a consequence of the secretory event and that secretion does not itself cause a decrease in input resistance.

Acetylcholine↗

PubMed1983

Origin of the decline in pancreatic amylase secretion with time in vitro.

The release of alpha-amylase (EC 3.2.1.1) into the medium bathing strips of rabbit pancreas decreased gradually with time in vitro so that after 3 h of incubation the secretory rate was only 20% of that seen initially. This decrease was not the result of an intrinsic loss of cellular responsiveness or viability, because a cholinergic agonist was still able to augment amylase secretion from the tissue after this incubation period to the same, or greater, degree than from "fresh" tissue. Rather, the inhibition of secretion was apparently due to the release of a nondialyzable substance from the tissue, which appears to be amylase itself. The observations that led to this conclusion are 1) replacing the bathing medium every 30 min eliminated the time-dependent decrease in enzyme secretion, 2) this replacement was ineffective if the tissue was separated from the medium by a dialysis membrane, and 3) adding an excess of the amylase substrate glycogen to the medium also prevented the falloff in amylase secretion, the effect of glycogen presumably being due to the formation of an enzyme-substrate complex in the medium. These observations are consistent with other observations that demonstrate that the rate of enzyme secretion by the pancreas depends on the rate of removal of product from the site of its secretion [Isenman and Rothman, Proc. Natl. Acad. Sci. USA 74: 4068-4072, 1977; Isenman and Rothman, Science 204: 1212-1215, 1979; Ho and Rothman, Am. J. Physiol. 242 (Gastrointest. Liver Physiol. 5): G32-G39, 1982].

Amylases↗

PubMed2001

Polymorphism in rice amylases at an early stage of seed germination.

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.

Germination↗

PubMed1991

Effect of glycosylation of bacterial amylase on stability and active site conformation.

With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site.

Amylases↗

PubMed1986

Catalytic flexibility of glycosylases. The hydration of maltal by beta-amylase to form 2-deoxymaltose.

Crystalline, alpha-glucosidase-free sweet potato beta-amylase was found to catalyze hydration of the enolic bond of maltal (alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-glucal) to form 2-deoxymaltose (alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-glucose). The reaction at pH 5.0 showed Vmax 0.082 mumol/min/mg and km 94.5 mM. An exceptionally large solvent deuterium isotope effect, VH/VD = 8, was observed from pH(pD) 4.2 to 5.4; and at pH(pD) 5.0 the effect was found to be directly related to the mole fraction of 2H. The hydration product, isolated from a beta-amylase/maltal digest in acetate-d4/D2O buffer (pD 5.4) was identified through its 1H NMR spectrum as alpha-D-glucopyranosyl-(1----4)-2-deoxy-D-[2(a)-2H]glucose. beta-Amylase in 2H2O thus catalyzes deuteration of the double bond of maltal from a direction opposite that assumed for protonation of the glycosidic oxygen atoms of starch chains and maltosaccharides. This finding confirms the functional flexibility of the enzyme's catalytic groups first demonstrated in studies of the reactions catalyzed with alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950). A possible mechanism of the maltal hydration by beta-amylase involves protonation of substrate from above as the first and rate-limiting step, followed by formation of a transient carbonium ion-enzyme intermediate. Although other possible mechanisms cannot be ruled out, it is clear that this hydration reaction differs from reactions catalyzed with amylaceous substrates and with alpha- and beta-maltosyl fluoride. The ability of beta-amylase to catalyze different types of reactions with different substrates is discussed with respect to observations with other enzymes that, likewise, strongly support the view (Hehre et al.) that the catalytic groups of glycosylases in general may be functionally flexible beyond requirements of the principle of microscopic reversibility.

Amylases↗

PubMed1977

Direct spectrophotometric determination of alpha-amylase activity in salive, with p-nitrophenyl alpha-maltoside as substrate.

We describe a simple, direct kinetic method for determination of salivary alpha-amylase (1,4, alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1). The assay makes use of a well-defined substrate, p-nitrophenyl alpha-maltoside, which is hydrolyzed by alpha-amylase to a chromogenic product, p-nitrophenol. Activity is determined by directly monitoring the increase in absorbance of the reaction mixture. Amylase activity can be defined in international (IUB) units of micromoles of product/min per liter of saliva. For 22 healthy subjects, the mean +/- SD of amylase activity in mixed saliva was 2.77 +/- 1.12 U/liter. Activity and instrumental response were linearly related over the entire range tested (0.224 to 11.90 U/liter). The within-run precision (CV) over this range was better than 3% for all but the lowest activities. Values obtained with this assay correlate well with those obtained with a modified Nelson-Somogyi saccharogenic method (r = 0.979). The precision and simplicity of this assay suggest that it is the method choice for determining amylase activity in human saliva.

Amylases↗

PubMed1979

[Alpha-amylase determination in acute pancreatitis: selection of a reference standard].

It has been investigated which of the amylase determinations agrees most closely with the clinical diagnosis in a group of patients with acute pancreatitis and in a group with other diseases producing amylase elevation. By measuring the amylase in a urine specimen related to its creatinine concentration fewer values within the range of reference in patients with pancreatitis and also fewer falsely elevated values in the second group were observed when compared to amylase in plasma, urinary amylase activity per volume or the amylase/creatinine clearance ratio.

Acute Disease↗

PubMed1994

Changes in serum amylase level following hepatic resection in chronic liver disease.

OBJECTIVE: Factors that were likely to cause hyperamylasemia following hepatic resection were studied. DESIGN: Case-comparison study. SETTING: Institutional practice. PATIENTS: Seventy-one patients who underwent hepatic resection because of primary or secondary liver tumors and other benign disease, and who had no history of pancreatitis, were divided into four groups. Patients were divided into groups according to the presence or absence of underlying liver disease and the vascular occlusion methods used during hepatic resection (Pringle maneuver or the hemihepatic vascular occlusion technique). The Pringle maneuver was chosen for patients in whom the duration of liver parenchymal transection was expected to be short and/or the hepatic hilum had severe adhesion precluding the safe dissection of the hepatic artery and portal vein. MAIN OUTCOME MEASURES: Serum amylase levels were measured on the preoperative day and on postoperative days 1, 2, 4, 6, 7, 10, and 14. Preoperative liver function, operative blood loss, operative time, and vascular occlusion time were examined. RESULTS: Patients with chronic liver disease (CLD group) had high serum amylase levels during the preoperative and postoperative periods. Patients in the CLD group who underwent the Pringle maneuver (Pringle-L group) had significantly elevated postoperative serum amylase levels in comparison with their preoperative serum amylase levels. Pancreatitis developed in two patients in the Pringle-L group--one of them died. CONCLUSION: These results strongly suggest that prolonged complete occlusion of the portal vein for hepatectomy in patients with chronic liver disease has a serious influence on postoperative serum amylase levels.

Adult↗

PubMed1988

Ectopic production of salivary-type amylase by a IgA-lambda-type multiple myeloma.

A patient with IgA-lambda-type multiple myeloma who appeared to be secreting salivary-type amylase ectopically is reported. Both the patient's serum and urine contained high levels of amylase. Amylase activity in the supernatant of cultured myeloma cells, obtained from the patient's pleural effusion while he had malignant pleuritis, increased almost linearly from the time of cell seeding. The presence of IgA and S-type amylase in the myeloma cells was demonstrated cytochemically and by immunoelectron microscopy. These observations showed that amylase was produced by these human myeloma cells.

Amylases↗

PubMed2000

Demonstration of amylase from the zoophytophagous anthocorid Orius insidiosus.

To better understand facultative phytophagy in the zoophytophagous anthocorid, Orius insidiosus, tests for amylase were conducted and the enzyme was partially purified. Three activity bands were detected with polyacrylamide-starch gel electrophoretic analysis of amylase in O. insidiosus. The major amylase was found to have a mean isoelectric point (pI) of 4.53. The presence of amylase indicates the ability of O. insidiosus to use starch, a nutrient, derived from plants, either by direct ingestion or by ingestion of plant material from the digestive system of their prey. The presence of amylase suggests that these predators are more committed to plant feeding than other species of predators that lack this enzyme.

Amylases↗

PubMed2000

Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch.

An improved method, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

Amylases↗

PubMed2006

Structure and enzyme properties of Zabrotes subfasciatus alpha-amylase.

Digestive alpha-amylases play an essential role in insect carbohydrate metabolism. These enzymes belong to an endo-type group. They catalyse starch hydrolysis, and are involved in energy production. Larvae of Zabrotes subfasciatus, the Mexican bean weevil, are able to infest stored common beans Phaseolus vulgaris, causing severe crop losses in Latin America and Africa. Their alpha-amylase (ZSA) is a well-studied but not completely understood enzyme, having specific characteristics when compared to other insect alpha-amylases. This report provides more knowledge about its chemical nature, including a description of its optimum pH (6.0 to 7.0) and temperature (20-30 degrees C). Furthermore, ion effects on ZSA activity were also determined, showing that three divalent ions (Mn2+, Ca2+, and Ba2+) were able to enhance starch hydrolysis. Fe2+ appeared to decrease alpha-amylase activity by half. ZSA kinetic parameters were also determined and compared to other insect alpha-amylases. A three-dimensional model is proposed in order to indicate probable residues involved in catalysis (Asp204, Glu240, and Asp305) as well other important residues related to starch binding (His118, Ala206, Lys207, and His304).

Amino Acid Sequence↗