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PubMed1980

Amylase secretion in response to activation of different autonomic receptors in the rabbit parotid gland.

The contribution by different autonomic receptors to the amylase secretion from the parotid gland of the anaesthetized rabbit was studied as the response to various parasympathomimetic and sympathomimetic drugs. Amylase secretion by infusions of pilocarpine and parasympathetic nerve stimulation was low, but regularly higher in response to pilocarpine than to parasympathetic nerve stimulation. These effects were reduced to the same level by beta-adrenoceptor block indicating the presence and for pilocarpine also the release of catecholamines, probably from the adrenals. Isoprenaline injections produced a high amylase secretion, that was blocked by atenolol, indicating that predominantly beta 1-adrenoceptors were activated. Phenylephrine was without amylase secretory effects. By accepting isoprenaline maximum as maximum for sympathetically produced amylase secretion, a theoretical frequency-response relationship for amylase secretion by sympathetic nerve stimulation could be calculated: ED50 was 0.9 Hz. The results indicate that under experimental conditions in vivo there are certain differences between the rat and the rabbit parotid glands in the autonomic control not only of fluid, but particularly of amylase secretion.

Amylases↗
PubMed1985

Sympathetic stimulation of amylase secretion during a parasympathetic background activity in the rat parotid gland.

The amylase secretion in vivo was examined in the rat parotid gland. A comparison was made between individual stimulation of sympathetic and parasympathetic nerves and simultaneous activation of both nerves. When sympathetic stimulation was superimposed on a parasympathetic background activity, amylase secretion was elicited at a frequency far below the threshold for secretion of fluid, and increased in a frequency-dependent way. This augmented amylase secretion, seen when the two nerves were activated at the same time, gave an amylase output which far exceeded the sum obtained at individual nerve stimulation. The sympathetic amylase secretion obtained in a background of parasympathetic activity seemed to be dependent entirely on beta 1-adrenoceptors. When high sympathetic stimulation frequency (3-10 Hz) was used a reduction in salivary flow was seen, which was accompanied by a reduced amylase output. This effect was counteracted by alpha-adrenoceptor blockade. Isoprenaline, injected intravenously during an ongoing parasympathetic activity, was found to evoke an augmented amylase secretion in a similar way as sympathetic nerve stimulation.

Adrenergic alpha-Antagonists↗
PubMed2006

Endogenous signalling system involved in parotid gland adenosine A(1) receptor-amylase release.

AIM: In this study, we have determined signalling pathways involved in adenosine A(1) receptor (A(1) receptor)-dependent stimulation of amylase release in rat parotid gland. METHODS: Amylase release, binding and cyclic adenosine monophosphate (cAMP) assays, inositol phosphates (IPs) production and nitric oxide synthase (NOS) activity in the presence of cyclopentyl-1,3-dipropylxanthine (CPA) alone or in the presence of different inhibitory drugs were performed. RESULTS: The binding parameters of specific A(1) antagonist [(3)H]-cyclopentyl 1,3-dipropilxanthine ([(3)H]-DPCPX) in parotid gland membranes show a population of high affinity sites with K(d) (nm) 0.53 +/- 0.06 and B(max) (fmol mg(-1) protein) 122.6 +/- 10.2. CPA stimulation of A(1) receptor exerts an increase in amylase release, IPs accumulation, cAMP production and NOS activity. All these A(1) agonist effects were blocked by the A(1) receptor antagonist DPCPX. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), and adenylate cyclase, but not NOS, activities attenuated the CPA stimulatory effect on amylase release. The effect of CPA on amylase release significantly correlated with its action either on cAMP or on IPs accumulation. CONCLUSION: These results suggest that CPA activation of parotid gland A(1) receptor induces a stimulatory effect on amylase release associated with increased production of cAMP and IPs accumulation. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving CaM and PKC. The CPA stimulation of NOS does not appear to participate in amylase release.

Adenosine A1 Receptor Antagonists↗
PubMed2004

Comparison of amylase and lipase activities in serum and plasma of dogs.

BACKGROUND: Amylase and lipase activities are most often determined in serum, although heparinized plasma is more convenient to obtain and is used for many routine biochemical analyses. OBJECTIVE: The purpose of this study was to compare amylase and lipase activities in serum and plasma of dogs and to determine whether either specimen type is acceptable for analysis. METHODS: Serum and heparinized plasma were obtained from 101 randomly selected dogs and analyzed in parallel for alpha-amylase and lipase. Results were compared using Passing-Bablock regression, Bland-Altman difference plots, and correlation analysis. RESULTS: There was a high correlation between the results obtained from serum and those from plasma. Regressions (with 95% confidence intervals in parentheses) were as follows: lipase(plasma) = 0.984 (0.976/0.995) Chi lipase(serum) - 0.9 (2.9/0.7) (r =.999); a-amylase(plasma) = 1.003 (0.977/1.032) Chi alpha-amylase(serum) - 1.9 ( 20.7/23.3) (r =.991). Mean differences (serum - plasma) were 8 U/L and 4 U/L for lipase and alpha-amylase, respectively. Classification of results as normal or abnormal did not differ according to specimen type. CONCLUSION: In dogs, lipase and alpha-amylase activities can be determined with the same level of accuracy in serum and in heparinized plasma.

Amylases↗
PubMed1973

Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release.

1. The effects of acetylcholine upon the output of amylase, Ca(2+) efflux and membrane potential of pancreatic acinar cells have been measured in segments of mouse pancreas superfused in vitro.2. Amylase output was measured continuously using an on-line automated fluorimetric method; Ca(2+) efflux was monitored by measuring the release of (45)Ca(2+) from pre-labelled tissue; and intracellular recordings of acinar transmembrane potentials were obtained with glass micro-electrodes. In some experiments membrane potentials, and in others (45)Ca(2+) efflux, were measured concomitantly with amylase release.3. Acetylcholine depolarized the acinar cells, increased tissue (45)Ca(2+) efflux and raised amylase output, each with a similar dose-dependence, i.e. a maximal response at 10(-5)M, threshold =/< 10(-8)M, and ED(50) values of 0.7 x 10(-7)M, 0.5 x 10(-7)M, and 2 x 10(-7)M for depolarization, amylase release, and (45)Ca(2+) efflux, respectively.4. In response to acetylcholine both depolarization and (45)Ca(2+) efflux preceded or coincided with the increase in amylase output.5. Acetylcholine 10(-5)M and [K](0) 47 mM were without effect on (45)Ca(2+) efflux in the presence of atropine (3 x 10(-6)M) but pancreozymin (0.3 u./ml.) still elicited a marked increase in (45)Ca(2+) release.6. These results suggest that the stimulatory action of acetylcholine on the pancreatic acinar cell involves, sequentially, a specific receptor-activated increase in membrane permeability, depolarization, Ca(2+) mobilization and amylase release. These events are discussed in relation to the integrated mechanism of stimulus-secretion coupling.

Acetylcholine↗
PubMed1977

Secretion of fluid and amylase in the perfused rat pancreas.

1. The isolated rat pancreas was perfused with physiological salt solutions of varying composition. Flow of pancreatic juice and output of amylase during rest and after stimulation with pure secretin, pure cholecystokinin-pancreozymin (CCK-PZ), caerulein or acetylcholine (ACh) were measured. 2. Basal fluid secretion was abolished replacing perfusion fluid NA+ or Cl- by Tris+ or SO42- respectively. Readmission of Na+ or Cl- caused a transient increase above the normal control level of both fluid and amylase output. Exposure to K+-free solution severely reduced fluid output and K+ readmission resulted in a transient increase in secretory rate. 3. Maximal stimulation with ACh (10(-7) M), CCK-PZ (1-5 X 10(-10) M) or caerulein (10(-10) M) caused marked sustained fluid and amylase secretion. Maximal secretin stimulation (5-7 X 10(-9) M) caused marked sustained fluid but only a small sustained amylase secretion following an initial transient. 4. Under continuous secretin stimulation, replacement of the CO2/HCO3-buffered control fluid by a CO2/HCO3-free Tris buffered solution caused a sharp decrease in pancreatic juice flow. In the absence of extracellular CO2/HCO3-secretin did not evoke fluid or enzyme secretion. In contrast the effects of ACh, CCK-PZ or caerulein were independent on CO2/HCO3-. Monobutyryl cyclic AMP (10(-3) M) caused marked sustained fluid secretion and transient enzyme secretion. The effect was entirely dependent on the presence of CO2/HCO3-in the perfusion fluid. 5. Ouabain (10(-4)-10(-3) M) markedly inhibited both secretin- and caerulein-evoked fluid secretion while caerulein-evoked amylase secretion was hardly affected. Similar findings were made with K+-free solution. 6. The effect of maximal secretin stimulation on amylase secretion was greatly augmented in the presence of a maximally stimulating concentration of caerulein. The effects on fluid secretion of secretin and caerulein were simply additive. The effects of secretin on both amylase and fluid secretion, in the presence of caerulein, were entirely dependent on the presence of CO2/HCO3- in the perfusion fluid. 7. We conclude that two different fluid secretion processes occur in the rat exocrine pancreas. One stimulated by ACh and CCK-PZ, that is independent of extracellular CO2/HCO3- and another stimulated by secretin involving H+ or HCO3-transport. Only the effects of secretin seem to be mediated by intracellular cyclic AMP.

Acetylcholine↗
PubMed1979

Calcium and cyclic nucleotide interaction in secretion of amylase from rat pancreas in vitro.

1. Interaction of Ca2+ and cyclic nucleotides in stimulus-secretion coupling in rat pancreas in vitro was studied utilizing the divalent cation inophore A23187. phosphodiesterase inhibitors, cyclic nucleotides and cholera toxin. 2. Amylase secretion was increased by the ionophore in the presence of extracellular Ca2+ in a dose-dependent fashion. Activation of CCK-PZ receptors simultaneously with induction of amylase secretion by A23187 did not alter amylase secretion whereas theophylline or caffeine had effects additive to A23187. Dibutyryl cyclic AMP potentiated the effect of ionophore whereas dibutyryl cyclic GMP had no effect on basal or ionophore-induced amylase secretion. Cholera toxin by itself did not effect amylase secretion whereas it potentiated the effect of ionophore. 3. A23187 increased bidirectional fluxes of 45Ca and increased efflux of 45Ca in a fashion similar to CCK-PZ. Theophylline did not alter basal efflux of 45Ca. Dibutyryl cyclic AMP increased the basal efflux of 45Ca whereas, cholera toxin, dibutyryl cyclic GMP and sodium butyrate had no effect. 4. Theophylline increased basal cyclic AMP levels with a peak effect observed at 5 min. Combination of theophylline and ionophore did not lead to an increase in levels of cyclic AMP greater than that observed with theophylline alone. Cholera toxin increased cyclic AMP levels at 30 and 60 min of incubation. 5. Ionophore and CCK-PZ increased tissue cyclic GMP levels significantly greater than that obtained with theophylline alone. This effect was dependent on extracellular Ca2+. The effect of ionophore on tissue levels of cyclic GMP could be dissociated from its effect on 45Ca efflux and amylase secretion. 6. It is concluded from these studies that Ca2+ plays a predominant role in regulating amylase secretion with interactions occurring between Ca2+ and cyclic AMP and Ca2+ and cyclic GMP. It appears that by themselves cyclic AMP and cyclic GMP do not play a significant role in regulating enzyme secretion.

Amylases↗
PubMed1982

The effect of extracellular calcium deprivation on amylase secretion and 45Ca efflux from rat pancreas.

1. The role of extracellular Ca(2+) in pancreatic enzyme secretion and (45)Ca efflux evoked by acetylcholine (ACh) and caerulein has been assessed in the incubated, uncinate pancreas of young rats.2. In a medium containing 2.5 x 10(-3)m-Ca(2+), the maximal rates of amylase secretion evoked by optimal doses of each secretagogue were similar. However, the time courses of amylase release during prolonged stimulation of the gland were different.3. The time course of amylase secretion in response to an optimal dose of ACh (10(-5)m) was characterized by an initial rapid increase followed by a slow sustained rise. For caerulein (10(-8)m), an initial rapid rise was followed either by a plateau or slight decline in the rate of amylase secretion.4. Both secretagogues produced similar increases in the rate coefficient of (45)Ca efflux from the gland.5. With supra-optimal doses of the secretagogues, amylase secretion, but not the rate coefficient of (45)Ca efflux, was depressed.6. Reducing the extracellular Ca(2+) concentration did not have a marked effect on basal amylase secretion but inhibited the action of both secretagogues. When the Ca(2+) concentration was 10(-6)m or lower, these inhibitory effects were irreversible. Amylase secretion stimulated by ACh was more sensitive to extracellular Ca(2+) deprivation than that stimulated by caerulein, the concentration required for half-maximal secretion being about 9-fold greater for ACh.7. Decreasing the extracellular Ca(2+) concentration increased both the basal and stimulated rate coefficients of (45)Ca efflux.8. Our results support the hypothesis that pancreatic enzyme secretagogues act by releasing bound Ca(2+) from sites within the acinar cell. Furthermore, they suggest that the site utilized by ACh is more amenable to depletion, via changes in extracellular Ca(2+) concentration, than that employed by caerulein.

Acetylcholine↗
PubMed1982

Role of cyclic adenosine monophosphate in amylase release from dissociated rat pancreatic acini.

1. The effect of octapeptide of cholecystokinin-pancreozymin (CCK(8)), bethanechol, cholera toxin, glucagon and vasoactive intestinal polypeptide (VIP) on amylase secretion and lactic dehydrogenase (LDH) release from isolated rat pancreatic acini was studied.2. In isolated rat pancreatic acini, in the absence of theophylline in the medium, amylase secretion was increased by 65-78% with 10(-7) and 10(-6) M-cholera toxin. In the presence of theophylline, amylase secretion was increased by 43-56% with 10(-7) and 10(-6) M-cholera toxin following a 90 min incubation. No effect was observed in the presence of theophylline at 30 and 60 min. The effect of cholera toxin was potentiated by CCK(8) at 60 and 90 min.3. In the absence of theophylline in the medium, amylase secretion was increased by 81-118% with 10(-5) and 10(-4) M-glucagon and 86% with 10(-6) M-VIP at 60 min. In the presence of theophylline in the medium, amylase secretion was increased by 53-246% with 10(-9) to 10(-6) M-glucagon and 111-158% with 10(-7) and 10(-6) M-VIP respectively. The effect of glucagon and VIP was potentiated by CCK(8).4. Potentiation of the rate of amylase release due to glucagon (10(-5) M) and VIP (10(-6) M) occurred during the first 15 min of incubation.5. Release of LDH was not increased by any of these agents.6. It is concluded that cyclic AMP rise (due to cholera toxin, glucagon and VIP effect) increased amylase secretion from rat pancreatic acinar cells. This effect is less marked than in the guinea-pig pancreas and is potentiated by agents mobilizing cellular Ca(2+) (CCK(8) and bethanechol).7. These data indicate species-specific variation in the action of cyclic AMP in the pancreas.

Amylases↗
PubMed1989

Augmentation of secretagogue-induced amylase secretion in pancreatic acini of heat-exposed rats.

1. The effects of prolonged heat exposure on pancreatic exocrine secretion were investigated to evaluate the involvement of the insulin-pancreatic acinar axis. Rats were kept at 34 degrees C and a relative humidity of 40% for 2 weeks with or without insulin administration. Control rats were housed at the thermoneutral temperature of 25 degrees C. By using isolated acini, secretory function was examined at the cellular level. 2. Without insulin treatment, acinar amylase concentration, expressed per microgram of cellular protein, was increased by heat exposure, while trypsinogen concentration, expressed per microgram of acinar DNA, decreased. The ratio of acinar amylase to trypsinogen increased significantly from 2.82 to 5.69 by prolonged heat exposure. With insulin treatment, the heat-induced increase in amylase activity was lessened but the decrease in trypsinogen remained unchanged. The ratio was somewhat lessened to 4.66. 3. In acini from saline-treated rats, amylase release in response to varying concentrations of cholecystokinin octapeptide or carbamylcholine was significantly augmented by prolonged heat exposure. As a result, the dose-response curve shifted upwards. However, in acini from insulin-treated rats, the increase in secretory response was lessened, similar to the effect on acinar content. On the other hand, changes in trypsinogen release were not as notable as those in amylase release. The ratio of amylase to trypsinogen in pancreatic juice released by 100 pM-cholecystokinin octapeptide increased from 1.56 to 3.04 in saline-treated rats and from 1.60 to 2.34 in insulin-treated rats. 4. In heat-exposed, saline-treated rats the plasma concentrations of glucose and insulin were significantly elevated, while in insulin-treated rats these increases were lessened and the elevation of plasma insulin concentration was no longer significant. 5. It is suggested that heat exposure elevates resting plasma glucose concentration, probably because of decreased metabolic activity, which results in a concomitant rise in plasma insulin concentration. This hormonal change most likely is a major cause of augmentation of acinar amylase synthesis and of the resultant potentiation of stimulus-secretion coupling via an insulin-pancreatic acinar axis. Heat exposure modified pancreatic exocrine function in the opposite direction to what occurred in cold-exposed animals. It was confirmed that changes in pancreatic exocrine function via a modification of the insulin-pancreatic acinar axis can actually occur under normal physiological circumstances. From the nutritional point of view, this modification by ambient temperature should be taken into consideration.

Amylases↗
PubMed1991

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148.

Amino Acid Sequence↗
PubMed1994

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis.

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.

Amino Acid Sequence↗
PubMed1974

Transformation of Bacillus subtilis in alpha-amylase productivity by deoxyribonucleic acid from B. subtilis var. amylosacchariticus.

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.

Amylases↗
PubMed1996

The glucose effect and regulation of alpha-amylase synthesis in the hyperthermophilic archaeon Sulfolobus solfataricus.

An alpha-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and alpha-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of alpha-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35S]-alpha-amylase protein is also subject to the glucose effect. alpha-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of alpha-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for alpha-amylase production, while glutamate was the most derepressing. The pattern of regulation of alpha-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.

Enzyme Induction↗
PubMed2003

Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.

Amino Acid Sequence↗
PubMed1967

Comparison of the alpha-amylase of Bacillus subtilis and Bacillus amyloliquefaciens.

The alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) of Bacillus subtilis strain W23 is less negatively-charged than the alpha-amylase of B. amyloliquefaciens strain F, as determined by electrophoretic mobility in polyacrylamide gel at pH 8.6. The alpha-amylase of strain W23 is immunologically unrelated to the alpha-amylase of strain F, as judged by lack of cross-reaction in Ouchterlony immunodiffusion studies. The pH range of maximal activity for the enzyme of strain W23 was 5.7 to 6.7, with a maximum at 6.3. The pH range of activity for the alpha-amylase of strain F was 5.5 to 6.5, with a maximum at 5.9. No significant difference was found in the effect of temperature on the activity of the alpha-amylase of strain W23 and strain F. alpha-Amylase production by strain W23 occurs throughout the 7-hr growth period, whereas enzyme production by strain F does not begin until the culture enters the stationary phase of growth. The total amounts of enzyme produced by strains W23 and F after 7 hr of growth were 0.3 and 25.5 units/ml, respectively.

Amylases↗
PubMed1983

Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium.

Highly purified salivary alpha-amylase inhibited the growth of fresh isolates of Neisseria gonorrhoeae on GC agar base medium supplemented with 2% IsoVitaleX (BBL Microbiology Systems). Hydrolysis of starch in the medium by amylase resulted in a negative starch-iodine test. However, purified amylase did not inhibit gonococcal growth on agar plates that contained hemoglobin (chocolate agar). This effect was not caused by inhibition of amylase, since amylase activity was the same in the presence or absence of blood products. Moreover, survival of N. gonorrhoeae in buffered saline was not affected by amylase. These results suggest that amylase inhibited the growth of N. gonorrhoeae on GC agar base plates by hydrolyzing starch.

Bacteriological Techniques↗