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PubMed1984

Radioimmunoassay of human salivary amylase: cross-reactivity with human and porcine pancreatic amylase and other salivary proteins.

A radioimmunoassay (RIA) of human salivary amylase was developed. Human salivary and pancreatic amylases were purified by Sephacryl S-200 gel filtration and by cation-exchange chromatography. Human salivary amylase antibody, raised in New Zealand white rabbits, did not crossreact with other salivary proteins and there was also no crossreactivity with purified porcine pancreatic amylase. The antibody crossreacted with human pancreatic amylase to the extent of 25%. Amylase concentrations, estimated by RIA, in human saliva, serum, and urine were compared with enzymatic activity. Correlation of results obtained by the two techniques was best for estimation of amylase in saliva, least for serum and intermediate for urine. Amylase concentrations and enzymatic activity in stimulated parotid saliva were not correlated with flow rate of secretion. There was no correlation between amylase concentrations (and enzymatic activity) in parotid saliva and those found either in serum and urine. Amylase comprises approximately 5% of the total parotid salivary protein in humans.

Amylases↗
PubMed2003

Lipase and pancreatic amylase versus total amylase as biomarkers of pancreatitis: an analytical investigation.

OBJECTIVE: To evaluate the biomarkers of pancreatitis Colorimetric Lipase, Total Amylase and Pancreatic Amylase (immunoinhibition) assays on the Roche COBAS INTEGRA 700. RESULTS: Pancreatic and Total Amylase assays and Colorimetric Lipase showed excellent imprecision of 1.6 to 2.3% and linearity (slope = 0.94-0.99, y-intercepts-1 to +3 U/L, r = 0.999) over the range of 17 to 900, 35 to 880, and 21 to 150 U/L, respectively. There was an excellent correlation between Pancreatic and Total Amylase: Pancreatic Amylase = 0.99 (+/- 0.02) x Total Amylase-36(+/- 8) (n = 106, r = 0.97, p < 1 x 10(-5), y intercept p < 1 x 10(-5)). Colorimetric Lipase showed some correlation to Total and Pancreatic Amylase results: Colorimetric Lipase = 1.54 (+/- 0.16) x Total Amylase-81(+/- 37) (n = 100, r = 0.70, p < 1 x 10(-6), y intercept p = 0.03), and Colorimetric Lipase = 1.78 (+/- 0.15) x Pancreatic Amylase-50(+/- 29) (n = 99, r = 0.78, p < 1 x 10(-6), y intercept p = 0.09). CONCLUSION: We recommend running the more specific Pancreatic Amylase as biomarker of pancreatitis on the Roche COBAS INTEGRA.

Amylases↗
PubMed1997

Molecular cloning of bruchid (Zabrotes subfasciatus) alpha-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors.

alpha-Amylases are important digestive enzymes in weevils that infest starchy seeds, and plants have evolved proteinaceous alpha-amylase inhibitors (alpha AI) for protection. To gain a better understanding of the interaction between weevil alpha-amylases and alpha AIs, we cloned the alpha-amylase cDNA of Zabrotes subfasciatus larvae. Larvae of this bruchid infest seeds of cultivated varieties of the common bean, Phaseolus vulgaris, although the seeds contain high levels of an alpha AI. The alpha-amylase cDNA, called ZsAmy, encodes a mature protein of 466 amino acids with a signal peptide of 17 amino acids. This protein has 50-60% amino acid identity with the other five known insect alpha-amylases. Three amino acid residues known to be important for catalysis and three histidine residues involved in substrate binding are conserved in the derived amino acid sequence of ZsAmy. Expression of ZsAmy with a baculovirus vector in cultured insect cells resulted in the production of active alpha-amylase, alpha AI-1, the form of the inhibitor found in cultivated beans, does not inhibit larval or expressed bruchid alpha-amylase, but alpha AI-2, a form of the inhibitor found in certain wild bean accessions, does inhibit the larval, as well as the expressed bruchid alpha-amylase. These and other observations lead to the conclusion that ZsAmy encodes the major larval amylase of this bruchid species.

Amino Acid Sequence↗
PubMedDate not supplied

The changes of alpha-amylase activity in serum and different tissues of female rat during sex cycle--isoelectrofocusing studies of alpha-amylase.

BACKGROUND: It has been suggested that hormonal changes may influence alpha-amylase activity during sex cycle. The aim of this study was to evaluate these changes in serum and different tissues during sex cycle in female rats. MATERIAL AND METHODS: The animals were assigned to proestrus, estrus, and diestrus groups depending on vaginal smears. We measured the activities of alpha-amylase in the serum, liver, salivary glands, pancreas and ovary of female rats, serum level of calcium, rat luteinising hormone (rLH) and rat follicle stimulating hormone (rFSH). The serum and tissue amylases were also characterized by wide-range polyacrylamide gel isoelectrofocusing. RESULTS: Statistically significant changes of alpha-amylase activity were found only in the ovary. The activity of alpha-amylase raised from proestrus (mean 303 +/- 124 IU/g) through estrus (mean 157 +/- 123 IU/g) and declined in diestrus (mean 81.9 +/- 51.6 IU/g). There were no statistically significant changes in the serum, liver, salivary glands and pancreas. We found two isoenzymes of alpha-amylase: one peak of amylase, present in the serum, liver, salivary gland and ovary with the mean isoelectric point of ca 5.34 and another present in ovary and pancreas with mean isoelectric point of ca 8.32. The only tissue found to contain both isoamylases was the ovary. We did not find any correlation between serum calcium level and alpha-amylase activity in the serum and ovary. CONCLUSION: The pattern of changes of alpha-amylase activity does not depend on the type of isoamylase in the tissue, but probably on the tissue influenced by the sex cycle. The changes of alpha-amylase activity are not mediated by calcium, they seem to result primarily from the profile of sex hormones affecting directly the target tissues.

Animals↗
PubMed1980

Functional significance of amylase polymorphism in Drosophila melanogaster. III. Ontogeny of amylase and some alpha-glucosidases.

Changes in amylase (E.C. 3.2.1.1), maltase (E.C. 3.2.1.20), sucrase, and PNPGase activities in relation to changes in wet weight and protein content were studied during the development of larvae and adult flies from two strains of Drosophila melanogaster, homozygous for different amylase alleles. All alpha-glucosidase activities increase exponentially during a large part of larval development, parallel to the increase in weight, and drop at the end of the third instar. Amylase activity of the Amy1 strain follows the same pattern. In contrast, amylase activity of the Amy4,6 strain continues its exponential increase longer. In the third larval instar amylase activity in the Amy4,6 strain becomes much higher than in the Amy1 strain. During the first hours of adult life amylase activity of the two strains does not differ. Then Amy4,6 activity starts to rise and becomes much higher (4-5 times) than Amy1 amylase activity, which remains approximately constant. All adult enzyme activities are much higher than in larvae. Comparison of enzyme activity of amylase and alpha-glucosidases in larvae and adults confirms that differences in amylase activities can become important only when starch is a limiting factor in the food.

Amylases↗
PubMed1998

A novel strategy for inhibition of alpha-amylases: yellow meal worm alpha-amylase in complex with the Ragi bifunctional inhibitor at 2.5 A resolution.

BACKGROUND: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin. CONCLUSIONS: The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Amino Acid Sequence↗
PubMed2001

Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments.

The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.

Binding, Competitive↗
PubMed2002

Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases.

Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, beta-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive alpha-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six different alpha-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, gamma-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to different modes of inhibition and different specificity profiles against diverse alpha-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own alpha-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has benefited from the recent determination of many structures of alpha-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of alpha-amylase inhibition. The continuing discovery of new classes of alpha-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect alpha-amylases, plant alpha-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.

Animals↗
PubMed1988

Amylase expression in human parotid neoplasms: evidence by in situ hybridization for lack of transcription of the amylase gene.

Salivary alpha-amylase (EC 3.2.1.1) is the major protein component of human parotid gland secretion. We studied amylase gene structure and expression in tissue from a series of normal and neoplastic parotid glands by Southern blot analysis, in situ hybridization, and immunohistochemistry. Thirty-two tumors were examined. Southern blot analysis of DNA extracted from a Warthin tumor, an adenoid cystic carcinoma, and a mucoepidermoid carcinoma showed no evidence of structural rearrangement of amylase genes. Eleven parotid Warthin tumors were negative for amylase protein and mRNA by immunocytochemistry and in situ hybridization. One pleomorphic adenoma in the group of 10 examined showed focal staining for amylase protein, although amylase mRNA could not be demonstrated in the same population of cells by in situ hybridization in serial tissue sections. Five mucoepidermoid carcinomas and three acinar cell carcinomas were devoid of amylase protein and mRNA. Normal parotid tissue obtained from all patients studied revealed abundant acinar cell amylase mRNA and protein. In situ hybridization, in conjunction with immunocytochemistry, allows precise cellular localization of mRNA and protein, thereby establishing the site of production of specific transcripts. We conclude that the interruption in amylase gene expression in parotid gland neoplasms occurs at the transcriptional level.

Amylases↗
PubMed1986

Separation and characterization of four different amylases of Entamoeba histolytica. II. Characterization of amylases.

Purified E. histolytica amylases III to VI were characterized by their hydrolytic behaviour towards 4-nitrophenyl alpha-malto-oligosaccharides, malto-oligosaccharides, amylose, amylopectin, glycogen and Y-cyclodextrin. The influence of specific inhibitors on the amylase activity of E. histolytica was examined and compared with typical alpha- and beta-amylases. Amylases III and IV showed alpha-glucosidase and glucosyltransferase activity by cleaving terminal non-reducing glucose from pNPG1 (III, IV) and pNPG2 to pNPG7 (III). Both enzymes were able to cleave malto-oligosaccharides and glucopolysaccharides to a large number of malto-oligosaccharides. Also transglucosidation reactions were observed, but maltose was not hydrolysed. Amylase V showed exoamylase-like properties by preferentially cleaving maltose units from the non-reducing end of synthetic and biogenic malto-oligosaccharides by a multiple-attack mechanism. Amylase VI was characterized as an alpha-amylase, showing great similarities with porcine pancreatic alpha-amylase in the hydrolysis pattern of 4-nitrophenyl alpha-malto-oligosaccharides and glucopolysaccharides. With biogenic malto-oligosaccharides amylase VI showed a transglucosidation reaction.

Amylases↗
PubMed1990

[Misleadingly high amylase and pancreatic amylase activity in the plasma of patients with macroamylasemia].

A man aged 68 years with choledocholithiasis and cholangitis, with no clinical signs suggestive of acute pancreatitis and with a low excretion of amylase in the urine, showed persistent hyperamylasaemia which appeared to be caused by macroamylasaemia. The macroamylase (an IgA-lambda-amylase complex) accounted for nearly all (90%) of the amylase activity in the serum. The activity of pancreatic amylase in serum, determined by an immunoinhibition test which selectively blocks salivary amylase activity, constituted 99% of the amylase activity in serum (normal reference range 19-71). We showed, however, that complexed salivary amylase is not inhibited in the test, resulting in a falsely-increased activity of pancreatic amylase in serum. We conclude that macroamylasaemia can lead to a clinically misleading increase in the activity of pancreatic amylase in serum.

Aged↗
PubMed1984

Automated measurement of amylase isoenzymes with 4-nitrophenyl-maltoheptaoside as substrate and use of a selective amylase inhibitor.

We automated a kinetic procedure for determining amylase isoenzymes in serum and urine samples. We used 4-nitro-phenylmaltoheptaoside as substrate and a selective amylase inhibitor with the Abbott-VP bichromatic system. By use of the maximum differences between pancreatic (P) and salivary (S) amylase activities remaining after inhibition by the selective inhibitor and by use of the linear range, a one-point standard method for calibration is proposed for determining amylase activities between about 50 and 1500 U/L when the P/S ratio exceeds 0.2. Results correlated well with those by electrophoresis and the Phadebas method (r = 0.99 for both pancreatic and salivary amylase). Reproducibilities (CVs) were 1.5% to 5.5% for pancreatic amylase and 1.4% to 3.3% for salivary amylase in serum, 0.8% to 2.0% for pancreatic amylase and 0.8% to 2.3% for salivary amylase in urine.

Age Factors↗