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PubMed1983

Localization of the two free thiol groups in the porcine pancreatic alpha-amylase I sequence.

Porcine pancreatic alpha-amylase I, a single 496 residue long polypeptide chain, contains 5 disulfide bridges and 2 free -SH groups. The conditions for specific blocking of native amylase either with radioactive N-ethyl maleimide or with labeled iodoacetic acid were determined. Under these conditions 2 moles of blocking reagent are incorporated per mole of amylase. [14C]-S-succinimido amylase was cleaved by CNBr and the resulting peptides were purified. Only one of them the CNBr 2 + 3 peptide (178 residues) was found labeled. Ts1 a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation Ts1A, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of Ts1A yielded 2 equally labeled peptides: C1 (16 residues) and C2 (6 residues). Automated sequencing of both peptides and counting of the PTH-amino acids shows that the free cysteines are only 15 residues apart in the sequence.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of the promoter and NH2-terminal signal peptide region of Bacillus subtilis alpha-amylase gene cloned in pUB110.

The nucleotide sequence of the promotor and NH2-terminal signal peptide region of the alpha-amylase gene derived from the alpha-amylase hyperproducing strain B. subtilis NA64 was determined. DNA sequences of the NH2-terminal region of the mature alpha-amylase, 41 amino acid residues of the signal peptide, a Shine-Dalgarno sequence (AGGAG), a potential RNA polymerase recognition site (TTGAAA), and a potential Pribnow box (AAGTAA) were identified. The DNA sequence was quite different from that of the alpha-amylase gene of B. amyloliquefaciens.

Amino Acid Sequence↗
PubMed1978

Cancer, pancreatitis, and the detection of the isoenzymes of DNAase, RNAase and amylase.

Substrate films of starch, RNA, and DNA were used to identify the isoenzymes of amylase, RNAase, and DNAase found in human ductal pancreatic juice subjected to isoelectric focusing. The pancreatic secretions from 15 patients were shown to contain as many as four isoenzymes of RNAase; the two major forms had isoelectric points of 7.87 and 7.52, and the two minor forms, of 7.25 and 6.90. Six DNAase bands were detected; the major bands had pI values of 4.86 and 4.79, and sometimes appeared as one band. The minor bands had pI values of 5.08, 5.00, 4.68, and 4.58. Purified bovine DNAase I, analyzed similarly, showed four bands (5.29, 5.19, 5.04 and 4.96). Nine isoenzymes of alpha-amylase were observed in the secretions from 15 patients. The major alpha-amylase isoenzyme had a pI value of 6.84 in 14 patients and of 7.04 in 1 patient. Secondary bands were seen with pI values of 6.23, 6.53 and 6.69. Additional isoenzymes were found with pI values of 7.16, 6.39, 6.00 and 5.78. The amylase isoenzyme with a pI value of 6.39 was found in 7 of the 8 patients with a normal pancreas or carcinoma of the pancreas, and in only 1 of 7 patients with acute or chronic pancreatitis.

Amylases↗
PubMed1981

Effect of albumin on amylase assay using blue starch polymer.

The nature of the stimulation by albumin of amylase activity determined by use of blue starch polymer was studied. The binding of albumin to the starch polymer was responsible for the stimulation of amylase activity, since the stimulation was observed with the albumin-bound blue starch polymer as substrate irrespective of the presence or absence of free albumin in the reaction mixture. When the albumin-bound blue starch polymer was hydrolysed by amylase, the blue oligosaccharide fragments were released with the albumin attached. These oligosaccharide fragments were about 1.3 times larger in average molecular size than the fragments released in the absence of albumin, suggesting that the apparent stimulation of amylase by albumin, (about 1.3 times) is due to the liberation of the larger oligosaccharide fragments.

Amylases↗
PubMed1981

Serum alpha-amylase isozymes in mumps: estimation of salivary and pancreatic isozymes by isoelectric focusing.

Serum alpha-amylase isozymes were separated into three major isozymes by thin-layer gel isoelectric focusing and detected by a starch-iodine zymogram procedure. Of the three groups of isozymes, one (S isozyme) corresponded to a salivary specific form, one (P isozyme) to a pancreatic specific form and the third (SP isozyme) to isozymes of similar isoelectric point common to both secretions. The levels of total alpha-amylase and of these three isozymes were estimated in the sera of 54 patients with mumps. Total alpha-amylase and salivary isozyme concentrations were greatly increased in the sera of all patients compared with controls. Pancreatic isozyme concentrations however, were only slightly increased and did not correlate with clinical pancreatitis. Indeed, in patients with mumps associated with pancreatitis, meningoencephalitis or orchitis, levels of total serum amylase, although higher than controls, were lower than levels in patients who presented solely with mumps sialadenitis.

Adolescent↗
PubMed1980

Tissue-specific expression of mouse-alpha-amylase genes: nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland.

We have determined the nucleotide sequence of two different mouse alpha-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively are the major alpha-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5' noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict alpha-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland alpha-amylase mRNA is not initiated at the AUG codon nearest the 5' terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.

Amino Acid Sequence↗
PubMed1985

Affinity chromatography of mustard beta-amylase on starch columns.

An affinity chromatography method for purification of beta-amylase from cotyledons of white mustard seedlings (Sinapis alba L.) is described. beta-Amylase is bound to starch column, while other contaminating proteins are eluted with the binding buffer. The bound beta-amylase is eluted by including dextrin (1%, w/v) in binding buffer. This method yielded a homogeneous preparation of beta-amylase enzyme, which migrated as a single polypeptide band in SDS gel electrophoresis.

Amylases↗
PubMed2026

Design, synthesis and biological evaluation of hydroxybenzothiazole-linked benzothiazole/benzoxazole conjugates as potent dual α-amylase and α-glucosidase inhibitors.

The current study focuses on the synthesis and evaluation of novel Hydroxybenzothiazole-Linked Benzothiazole/Benzoxazole Conjugates to target Diabetes Mellitus (DM) by inhibiting α-amylase and α-glucosidase. Spectroscopic methods, including 1H and 13C NMR spectroscopy, were employed to confirm the structures of newly synthesized conjugates. The findings of in-vitro analysis displayed that the synthesized derivatives inhibited α-amylase and α-glucosidase enzymes with IC50 values ranging from 3.65 ± 0.20 μM to 32.15 ± 3.20 μM on α-amylase and 5.92 ± 0.80 μM to 35.60 ± 3.40 μM on α-glucosidase, in contrast to the reference drug Acarbose (α-amylase IC50 = 8.25 ± 0.80 μM; α-glucosidase IC50 = 10.75 ± 1.10 μM). Among the series 9a-9f and 10a-10f, analogs 10 f, 10b, 9b, and 9e displayed superior anti-diabetic activity compared to the reference drug Acarbose. The inhibitory activity of these conjugates can be attributed to their favorable and stable interactions with critical amino acid residues of targeted enzymes, as revealed through molecular docking analysis. ADMET predictions and drug-likeness evaluations showed favorable pharmacokinetic features, while DFT investigations revealed electronic insights related to bioactivity. Experimental outcomes and in silico support display that these potent Hydroxybenzothiazole-Linked Benzothiazole/Benzoxazole Conjugates were comparable to an existing diabetic mellitus inhibitor while conserving an acceptable safety profile, specifying potential for further therapeutic development and optimization against diabetic Mellitus.

Benzothiazoles↗
PubMed1978

An improved assay method for amylase activity using an amylodextrin fraction as substrate.

A rapid and simple method for determination of amylase activity, by using a fraction of amylo-oligosaccharide (amylodextrin, 1) as substrate, is described. The sample solution is incubated with a solution of 1 and the substrate consumed is estimated by measuring the difference in absorbance at 460 nm. The Km value of 1 is about half that of starch. The homogeneity of 1 in its chemical structure and molecular weight facilitated the specification of amylase units according to the international definition. This procedure was applied for assay of human-serum amylase with excellent reproducibility. This method did not require a large dilution factor, as the standard curve showed a linear relationship over a wide range of amylase concentrations.

Amylases↗
PubMed1978

A purification of Helix pomatia alpha amylase involving a novel affinity-binding procedure.

Shellfish glycogen was cross-linked by treatement with cyanogen bromide followed by 1,6-diaminohexane. The resulting, insoluble product efficiently adsorbed Helix pomatia alpha amylase [(1 linked to 4)-alpha-D-glucan glucanohydrolase] from crude solutions of the enzyme at 0 degrees, but only poorly at higher temperatures. A method was developed for the purification of Helix pomatia alpha amylase involving formation of an enzyme-adsorbent complex in the cold and recovery of the alpha amylase by suspending the washed complex in buffer at 37 degrees. After chromatography of the desorbed alpha amylase on a column of Bio-Gel P-60, the enzyme was homogenous as judged by poly(acrylamide)-gel electrophoresis. An overall purification of 360-fold was achieved with a recovery of 35%.

Adsorption↗
PubMed1982

Immunoreactive trypsin, alpha-amylase and lipase in serum--is there an age-dependence?

A strong age-dependence of IR trypsin was found in 424 healthy controls suggesting the need for age-matched reference values. A possible relation of other exocrine pancreatic enzymes like alpha-amylase and lipase to age was investigated in 185 apparently healthy persons undergoing a preventive health check. Only alpha-amylase showed a weak correlation with age, but not lipase. Both enzymes correlated with IR trypsin. Whilst alpha-amylase and lipase activities were within the normal range, approximately 25% of IR-trypsin concentrations were above age-matched control values. It is suggested that these apparently healthy persons already have disease of the exocrine pancreas, and IR trypsin is a more sensitive diagnostic tool for screening than alpha-amylase or lipase.

Adolescent↗
PubMed1998

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution.

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.

Amino Acid Sequence↗
PubMed1980

Sequence of the N-terminal half of Bacillus amyloliquefaciens alpha-amylase.

Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 3.2.1.1), which is commercially supplied as 'Bacillus subtilis alpha-amylase' does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).

Amino Acid Sequence↗
PubMed1983

The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins.

A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7.

Amylases↗
PubMed1982

Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence.

We describe a secretion vector system for introducing foreign genes into Bacillus subtilis. We constructed secretion vectors from the plasmid pUB110 and the promoter and signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens. Foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. Demonstrating secretion of a foreign gene product from Bacillus, we here report that the Escherichia coli beta-lactamase gene, devoid of its own signal sequence coding region, can be expressed in B. subtilis by the aid of the secretion vectors so that greater than 95% of the enzyme activity is secreted to the growth medium. Efficient secretion of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is observed if the complete signal sequence coding region of the alpha-amylase gene precedes the beta-lactamase structural gene. However, an incomplete alpha-amylase signal peptide lacking the six carboxy-terminal amino acid residues does not promote secretion of the fused beta-lactamase, which remains unprocessed and cell-associated.

Amino Acid Sequence↗
PubMed1983

Enzyme immunoassay for alpha-amylase.

A sensitive sandwich enzyme immunoassay has been developed for quantitation of alpha-amylase in pig pancreas. The alpha-amylase-antibody conjugate was prepared from horseradish peroxidase coupled with antibodies to hog pancreas alpha-amylase. With this method the measuring range is 1-10 ng/ml and the detection limit is 0.2 ng/ml. The amount of pig pancreatic amylase was measured to be 6.21 +/- 2.05 mg/g of tissue. The enzymatic activity determination and immunoassay were compared in the material studied.

Amylases↗
PubMed1983

Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha-amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the alpha-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-beta-N-acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor II) forms, in addition to the mature form of alpha-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of alpha-amylase.

Acetylglucosaminidase↗
PubMed1983

Location of SH groups along the polypeptide chain of soybean beta-amylase.

The five SH groups of soybean beta-amylase differ in reactivity toward SH reagents such as 2,2'-dithiopyridine (2-PDS), monoiodoacetate and N-ethylmaleimide (NEM). They were designated as SH1, SH2, SH3, SH4, and SH5, in order of their reactivity except for the two buried SH groups, SH4 and SH5. The location of the five SH groups along the polypeptide chain was determined by specific cleavage at the amino side of their cyanocysteine residues which were formed by converting SH to SCN groups by cyanide after modifying the SH groups with 2-PDS. The selective modification of SH groups was achieved as follows: SH1 reacted with 2-PDS at low and high ionic strength, while SH2 reacted only at high ionic strength. SH2 and SH3 were also modified with 2-PDS using SH1-carboxymethylated soybean beta-amylase. The buried SH groups, SH4 and SH5, were modified with 2-PDS under the denaturation conditions after the reactive SH groups, SH1, SH2, SH3, were irreversibly blocked with NEM. On the other hand, the five SH groups were cyanylated with [14C]cyanide or with 2-nitro-5-thiocyanobenzoic acid (NTCB) for the cleavage at all five SH groups. The molecular weight estimation of derivatives of cleaved soybean beta-amylase by SDS-gel electrophoresis showed that the five pairs of fragments (Mw 50,000 & 6,500, 47,000 & 8,000, 38,000 & 18,000, 35,000 & 23,000, and 31,000 & 25,000) were identified with the fragments formed by cleavage at SH1, SH2, SH3, SH4, and SH5, respectively. By considering fragments incorporating 14C (Mw 47,000, 35,000, 25,000, 18,000, and 6,500), the fragments were aligned along the polypeptide chain of soybean beta-amylase, in order from the N-terminus as SH2, SH5, SH3, SH4, and SH1. This order is supported by estimating the molecular weight of fragments formed by high-yield cleavage using NTCB and by analyzing the COOH-terminal residues of the fragment cleaved at SH2.

Amylases↗