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The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.
To understand the cellular origin and mechanism of gene expression in amylase-producing cancers, the phenotyping of amylase isogenes by the polymerase chain reaction and restriction-fragment-length polymorphism using restriction endonucleases TaqI, DdeI, HinfI, and AfaI were performed for 3 amylase-producing lung adenocarcinomas, 16 lung cancers without hyperamylasemia, other human malignant neoplasms, cultured cell lines, and normal tissues. In addition, amylase mRNA transcripts were semi-quantified by the limited polymerase chain reaction. Amylase mRNA transcripts were detected in all of the tissues examined. The AMY1 gene (salivary type) was exclusively and highly expressed in the salivary glands and the amylase-producing lung adenocarcinomas. Coexpression of the AMY1 gene and AMY2 gene (pancreatic type) was observed in most of the lung cancers without hyperamylasemia, lung tissue, and cells scraped from the tracheal epithelium, thyroid, and female genital tract (ovary, fallopian tube, and uterus cervix), while minimal levels of mRNA transcripts of the AMY2 gene were detected in other malignant neoplasms, various normal tissues, and the cultured cell lines. All mRNA transcripts identified as being those of the AMY2 gene were further identified as being from the AMY2B gene except for the transcripts from the pancreas, in which the AMY2A gene and AMY2B gene were coexpressed. On the basis of these results, the clinical occurrence of amylase-producing cancer likely relates to the tissues expressing the AMY1 gene, while the AMY2B gene, which evolutionarily is the oldest gene among human amylase isogenes, is constitutively expressed in various tissues.
The alpha-amylase molecule secreted from the scutellar tissues of rice seedlings bears asparagine-linked oligosaccharides which include both (modified) complex-type and high-mannose-type structures. On the basis of their sensitivity to endo-beta-N-acetylglucosaminidase (Endo-beta-H), they are designated as R and S types. When labeled with [3H]fucose a typical R-type alpha-amylase is labeled. By contrast, [3H]mannose-labeled alpha-amylase can be partly digested by Endo-beta-H; hence, it contains both R and S molecules. The role of the Golgi complex in the post-translational oligosaccharide maturation of alpha-amylase was explored by use of the carboxylic ionophore, monensin (10(-7)M), a known perturbant of the structure and function of the Golgi complex. The monensin sensitivity of alpha-amylase transport and acquisition of terminal sugars as well as the morphologic consequences of monensin treatment point to a similarity between the Golgi complex of plant and animal cells. In order to elucidate the relationship between the secretion of two different forms of alpha-amylase and the partial inhibitory effect exerted by monensin, the possible role of Ca2+ in the secretory pathway was examined. The secretion of the R form was stimulated by Ca2+, whereas that of the S form was not affected by the external concentration of Ca2+. In pulse-chase experiments, we found that R-type alpha-amylase accumulates intracellularly under Ca2+-free conditions. These results indicate that there is both Ca2+-dependent and Ca2+-independent secretion of alpha-amylase in the rice scutellar epithelium cells.
alpha-Amylases from germinated maize, oats, rice, and sorghum were isolated by glycogen precipitation and hydrophobic interaction chromatography. Several methods were used for the detection of glycoproteins, including barley alpha-amylase isozymes purified as previously described and using the rice alpha-amylase as a positive control for glycosylation. Affinoblotting using concanavalin A, immunoblotting using a xylose-specific serum which reacts with complex N-linked glycans, and endo-beta-N-acetylglucosaminidase H treatment of amylases gave negative results for maize, oats, sorghum, and barley. However, after deglycosylation with trifluoromethanesulfonic acid, the molecular weight of one maize alpha-amylase constituent was clearly decreased. The same result was obtained after beta-elimination in mild conditions. Together these results indicated probable O-linked glycosylation of one maize alpha-amylase when barley, oats, and sorghum alpha-amylases did not appear to be glycosylated. Chemical deglycosylation of rice alpha-amylase resulted in the production of two polypeptides with different molecular weights.
A radioimmunoassay (RIA) for human pancreatic amylase has been developed for the determination of human serum amylase content. The assay was shown to be sensitive (7 ng/ml), reproducible and specific, but human pancreatic amylase and salivary amylase could not be distinguished by the antiserum used. In normal subjects, the mean concentration of amylase determined by the RIA was found to be 122.1 ng/ml (range: 55--250 ng/ml). A good correlation was observed between the concentration of amylase and its enzymatic activity in normal subjects. In some instances with high amylase activity, however, the rise in enzymatic activity was not accompanied by increasing amount of amylase content.
We describe a simple method to determine serum amylase isoenzyme activity with alpha-4-nitrophenyl-maltoheptaoside as substrate and the use of an amylase inhibitor. Day-to-day reproducibility (CV) was 2% for total amylase, 3-5% for pancreatic and salivary amylase; within-day precision was 1% for total amylase, 1-4% for pancreatic and salivary amylase. The concentrations of total, pancreatic and salivary amylase were determined in 169 sera obtained from healthy adults (82 men and 87 women). Total, pancreatic and salivary amylase concentrations in males were respectively 184, 105 and 66; in females 210, 97 and 92 U/l (mean). Our method is simple and rapid; our results agree well with those of other authors, who have used electrophoretic or blue starch methods.
Four alpha-amylase inhibitors, WRP24, WRP25, WRP26, and WRP27, were purified from wheat flour by preparative, reversed-phase high performance liquid chromatography. All have polypeptide molecular masses of about 14 kDa and are members of the cereal superfamily of protease and alpha-amylase inhibitors. Sedimentation velocity analysis indicated that WRP25 and WRP27 are monomeric proteins, whereas WRP24 is a dimer. WRP24 is identical in N-terminal amino acid sequence to the well characterized 0.19 dimeric inhibitor from wheat kernels. WRP25 and WRP26 differ in sequence from each other at only three positions and represent previously unseparated forms of the 0.28 wheat inhibitor. WRP27 is a previously uncharacterized inhibitor and is more similar in sequence to the 0.28 inhibitor than to the 0.19 inhibitor. WRP25 and WRP26 inhibited alpha-amylases from the rice weevil, red flour beetle, and the yellow meal worm, but did not inhibit human salivary alpha-amylase. WRP24 inhibited the human as well as the insect alpha-amylases, but inhibited one of the two rice weevil alpha-amylases much more strongly than the other. WRP27 was notable in that, of the enzymes tested, it strongly inhibited only the rice weevil alpha-amylases. We observed that the growth rate of red flour beetle larvae was slowed when purified WRP24 was included in the diet at a level of 10%. Addition of WRP24 to corn starch resulted in greater weight loss of red flour beetle adults than occurred on control diets. Our results support the hypothesis that these alpha-amylase inhibitors provide wheat seeds with a selective evolutionary advantage since the inhibitors can slow the growth of insect pests that attack cereal grains.
An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.
Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.
The relation between plasma and biliary amylase activity and their relationship to the functional state of the pancreas were studied in anesthetized rabbits. Repetitive intravenous injections of cholecystokinin resulted in a 25-fold rise in the secretion of amylase via the pancreatic duct, followed at first by a 50% increase in plasma amylase concentration and later by a 270% increase in biliary amylase concentration. There was then a gradual, roughly synchronous decline in both plasma and biliary values toward basal level despite a continued highly augmented rate of pancreatic ductal secretion. "Near-total" pancreatectomy completely abolished the effect. These observations are consistent with a cholecystokinin-induced basolateral secretion of amylase from pancreas into blood and its subsequent movement from blood into bile down a concentration gradient. The output of amylase in bile, however, was quite small and does not suggest that biliary transport of amylase has an important function either as a means of secreting and recycling digestive enzyme into the gut or as a major excretory pathway for circulating amylase in the rabbit.
Lingual amylase and lingual lipase, two digestive enzymes that are secreted from lingual serous glands (von Ebner's), were simultaneously purified from rat lingual serous glands with hydrophobic chromatography used as the final step. This method, previously developed for the purification of lingual lipase, includes homogenization of rat lingual serous glands, 100,000 g centrifugation, ammonium sulfate precipitation of proteins, and extraction of lipids with acetone at -20 degrees C, followed by hydrophobic chromatography on ethyl agarose or Agethane. Amylase was eluted after the elution of proteins that did not interact with the hydrophobic gel at pH 6.3. Lingual lipase was eluted with a solution containing micelles of taurodeoxycholate, monoolein, and oleic acid. Analysis of each of the purified enzymes by SDS-polyacrylamide gel electrophoresis revealed one band at Mr = 59,000 for amylase and one band at Mr = 51,000 for lingual lipase. Isoelectric focusing of amylase indicated a strong band at pI = 5.0 and two very faint bands at pI = 4.9 and 4.8, possibly isozymes or deamidated protein. Amino acid and hexosamine analyses were performed on the enzymes after electroelution from SDS-polyacrylamide gels. Both lingual lipase and lingual amylase had a high content of dicarboxylic (free and amide) amino acids. For lingual lipase and lingual amylase, the % molar ratios of aspartic acid/asparagine were 15.35 and 15.10, and the % molar ratios of glutamic acid/glutamine were 7.07 and 7.20, respectively. Lingual amylase was very similar to rat parotid, pancreatic, and mouse salivary amylases, except that it contained more proline (11.03% molar ratio).(ABSTRACT TRUNCATED AT 250 WORDS)
The two isozymes of pancreatic amylase in mouse strain YBR/Ki are encoded by closely linked genes which are independently regulated. We have isolated these two pancreatic amylase genes, Amy-2.1 and Amy-2.2, from a cosmid library of YBR/Ki genomic DNA and compared the nucleotide sequences of coding regions with the amino acid sequences of the protein isozymes. Transcripts of both genes were also isolated from a pancreatic cDNA library and partially sequenced. The results demonstrate that Amy-2.1 encodes the A1 isozyme of YBR/Ki pancreatic amylase, while Amy- 2.2 encodes the insulin-dependent B1 isozyme. Similarities of restriction maps and nucleotide sequences suggest that Amy-2.1 is closely related to the active Amy-2a gene previously isolated from strain A/J (Schibler, U., Pittet, A.-C., Young, R. A., Hagenbüchle, O., Tosi, M., Gellman, S., and Wellauer, P. K. (1982) J. Mol. Biol. 155, 247-266). Expression of Amy-2.2 may be limited to strain YBR/Ki. The inactive Amy-X gene from A/J (Schibler, U., Pittet, A.-C., Young, R. A., Hagenbüchle, O., Tosi, M., Gellman, S., and Wellauer, P. K. (1982) J. Mol. Biol. 155, 247-266) is apparently a null allele of Amy-2.2. An additional amylase gene from YBR/Ki has been identified as a pancreatic amylase pseudogene which diverged between sixteen and thirty-two million years ago. The pancreatic amylase subfamily in strain YBR/Ki thus consists of two active genes and one pseudogene. The low rate of amylase production in YBR/Ki pancreas, relative to that of other inbred strains, can be accounted for by the lower number of gene copies in this strain. Comparison of pancreatic amylase genes from different inbred strains provides evidence for several duplication and deletion events during the recent evolution of this chromosome region.
Gastrointestinal disease other than hepatobiliary and pancreatic disorders was associated with hourly rates of urinary amylase excretion above the limits of normal for control subjects (88 IU/hour compared with 69 IU/hour). In hepatobiliary disease, excretion rates of more than 88 but usually less than 190 IU/hour were sometimes found. Whilst rates of urinary amylase excretion were not helpful in the diagnosis of chronic pancreatitis or carcinoma of the pancreas, levels above 190 IU/hour were found in acute pancreatitis at a time when the serum levels were also diagnostic. After the acute episode the rate of urinary amylase excretion was moderately elevated for up to six days but did not reach diagnostic levels. Persistent elevation of serum amylase and lipase levels and hourly rates of urinary amylase excretion for more than six days suggested that a pseudocyst had developed. In acute pancreatitis the level of serum lipase was more frequently raised and persisted so for longer than either the serum or urinary amylase. Although the hourly rate of urinary amylase excretion is of little value alone, when performed in conjunction with evaluating the serum amylase and lipase it may provide useful additional evidence of pancreatic disease and it could be useful in the diagnosis of relapsing chronic pancreatitis.
Insulin-dependent diabetes mellitus type 1 is an autoimmune disease of pancreatic beta-cells with a certain genetic predisposition that is not yet clear. In spite of the confirmed association of diabetes mellitus type 1 with several HLA haplotypes it is considered that other loci must be involved for total genetic susceptibility to the disease. The relationship of insulin deficiency and decreased pancreatic amylase activity suggests that insulin itself is a direct activator of amylase gene expression. Endocrine pancreatic function was monitored by the indirect non-invasive method of urinary pancreatic amylase activity determination (expressed in percentage of total amylase activity) in diabetic children, their parents, healthy sisters and brothers, and in a separate group of women with diabetes type 1 of over 20 years duration. The incidence of hereditary amylase polymorphism variants in these subjects was also ascertained. Decreased pancreatic amylase activity in urine (under 58%) was found to be a characteristic trait in diabetics, and a susceptibility trait in asymptomatic family members. Normal pancreatic amylase activity (66.7 +/- 5.4%) is rare in diabetic patients type 1, but may be seen as a favourable prognostic trait, representing resistance to diabetic complications. The results support the suggestion that hereditary predisposition to the disease is inherited from the father rather than the mother, and that heterozygous amylase polymorphism variants protect their carriers against diabetes mellitus type 1.