Abstract:Alpha-Amylase (EC 3.2.1.1) was purified from the muscle and intestine of the parasitic helminth of pigs Ascaris suum. The enzymes from the two sources differed in their properties. Isoelectric focusing revealed one form of a-amylase from muscles with pl of 5.0, and two forms of amylase from intestine with pI of 4.7 and 4.5. SDS/PAGE suggested a molecular mass of 83 kDa and 73 kDa for isoenzymes of a-amylases from intestine and 59 kDa for the muscle enzyme. Alpha-Amylase from intestine showed maximum activity a… Show more
“…We also observed an increment in α-amylase activity with increased concentrations of CaCl 2 up to 25 mM, while the activity was not affected with subsequent increases. Similar results were found in α-amylases from other invertebrates [ 70 , 71 ], although in some insects high CaCl 2 concentration inhibited α-amylase activity [ 72 ]. In the case of lobster and other carnivorous crustaceans, calcium concentrations higher than in sea water (~10 mM) are expected in the digestive tract due to the ingestion of other crustaceans and mollusks with calcareous exoskeleton, and this appears to increase α-amylase activity in lobster up to 25 mM CaCl 2 .…”
Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
“…We also observed an increment in α-amylase activity with increased concentrations of CaCl 2 up to 25 mM, while the activity was not affected with subsequent increases. Similar results were found in α-amylases from other invertebrates [ 70 , 71 ], although in some insects high CaCl 2 concentration inhibited α-amylase activity [ 72 ]. In the case of lobster and other carnivorous crustaceans, calcium concentrations higher than in sea water (~10 mM) are expected in the digestive tract due to the ingestion of other crustaceans and mollusks with calcareous exoskeleton, and this appears to increase α-amylase activity in lobster up to 25 mM CaCl 2 .…”
Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
“…Amylase forms (isozymes) have been reported from other organisms. Three isozymes from Heterorhabditis bacteriophora (Mohamed 2004), two forms from Ascaris suum (Zoltoweska 2001) and one form from Thermus sp. (Shaw et al.…”
In the current study, midgut a-amylase from Sunn pest (Eurygaster integriceps Puton) (Hemiptera: Scutelleridae), one of the most serious pests of wheat and barley in the wide area of the Near and Middle East, West Asia, and many of the new independent states of central Asia, were purified and characterized. Amylase activity was detected in the midgut of the insects which were collected from both over-wintering sites during winter and feeding insects during spring. Amylase activities in the midgut of over-wintering and feeding insects were 5.71 and 3.43 U/mg protein, respectively. Initially, a native electrophoretic analysis of E. integriceps crude midgut extract showed that there are two major amylase forms in the midgut. Through the sequence of ammonium sulfate precipitation, first by gel filtration chromatography (Sephadex G-75), anion exchange chromatography (diethylaminoethylcellulose) and second by gel filtration chromatography, specific activity of a-amylase of E. integriceps increased 44-fold from approximately 3 to 133 U/mg protein. Analysis of purified amylases by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that these proteins had estimated molecular masses of 49 and 52 kDa. Optimum temperature was determined to be 30-40°C. The optimum pH value was 6.5 and the Kmapp for soluble starch was 0.54%.
“…α-Amylase in extracts from the two stages of A. simplex larvae were identified after PAGE on polyacrylamide gel prepared with 1% soluble starch (fig. 1), according to the procedure described by Żółtowska (2001a). Fig.…”
Section: Methodsmentioning
confidence: 99%
“…In nematodes, glycogen may be broken down by hydrolysis, amylases ( α-amylase and glucoamylase), or phosphorolysis, the process involving glycogen phosphorylase (Żółtowska et al , 2001, 2002). The hydrolytic glycogen degradation pathway in nematodes is controlled by α-amylase (Żółtowska, 2001a; Mohamed, 2004), an enzyme found in A. simplex . A preliminary characterization of α-amylase isolated from A. simplex larvae was published by Łopieńska et al (2001).…”
Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.
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