Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.     

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.     

Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions

The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo expression of UDP-N-acetylglucosamine: Alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-1) in Aspergillus oryzae and effects on the sugar chain of alpha-amylase

UDP-N-Acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man(5)GlcNAc(2). The N-linked sugar chain of alpha-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after beta-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan(5)GlcNAc(2) as a sugar chain of alpha-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of alpha-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.     

Enzymatic synthesis of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside as a substrate for human alpha-amylases.

Enzymatic modification at the nonreducing end D-glucosyl residue of p-nitrophenyl alpha-maltopentaoside was developed by using the transglycosylation of beta-D-galactosidase from Bacillus circulans. The enzyme regioselectively synthesized p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside (a yield of 12.0% based on the amount of p-nitrophenyl alpha-maltopentaoside added) on a preparative scale from lactose as a donor and p-nitrophenyl alpha-maltopentaoside as an acceptor. It revealed that the nonreducing end galactosyl group of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside did not prohibit the action of human salivary and pancreatic alpha-amylases. This derivative was shown to be very suitable as a novel substrate for analytical use of human alpha-amylase assay in serum through a conjugated reaction involving glucoamylase and alpha-D-glucosidase.     

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.     

A comparison of the modes of actions of human salivary and pancreatic alpha-amylases on modified maltooligosaccharides

The actions of three isozymes of human pancreatic alpha-amylase (HPA) on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo, an amino, or a carboxyl group at their first or penultimate glucopyranosyl residue from the non-reducing-end were examined. The results revealed that there was no difference in the actions of the three isozymes on the modified substrates and suggested the presence of five subsites (S3, S2, S1, S1', and S2') and a hydrophobic amino acid residue at subsite S3 in the active site of HPA. As compared with the action of human salivary alpha-amylase (HSA) on the same substrates, HPA had a tendency to release more phenyl alpha-glucoside from every substrate; however, an iodo, an amino, and a carboxyl group of the substrates had the same effects on the binding modes of the substrates to the active site of HPA as seen in the case of the salivary enzyme. This result indicates that the three-dimensional structures of the active sites of both alpha-amylases are quite similar except for some minor changes at subsites S3 and S2'.     

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.     

Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity

In the mechanism of hydrolysis of starch by alpha-amylases, a conserved water molecule bridging two catalytic residues has been implicated. In human salivary alpha-amylase (HSAmy), this water (W641), observed in many alpha-amylase structures, is part of a chain of water molecules. To test the hypothesis that W641 may be involved in the mechanism, Phe256 in the close vicinity was mutated to a Trp residue. X-ray structure of F256W complexed to 2-amino-2-(hydroxyethyl)-1,3-propanediol at 2.1A revealed that the water chain is disrupted. In the F256W structure exhibits a positional shift in His305, characteristic of alpha-amylase complex structures. Kinetic analysis, in comparison with HSAmy, revealed that the mutant exhibited a 70-fold decrease in the specific activity for starch and significantly reduced k(cat) (20-fold) and K(m) (4-fold) for maltoheptaoside. Collectively, these results suggest that W641 and the chain of water molecules may be critical for the alpha-amylase activity.     

Primary structure of human salivary alpha-amylase gene

A recombinant clone which covers the human salivary alpha-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary alpha-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene [Schibler et al., J. Mol. Biol. 155 (1982) 247-266].     

Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase

The mechanism of allosteric activation of alpha-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic alpha-amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride-dependent alpha-amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent alpha-amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent alpha-amylases.     

Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit-subunit interface of the human Rad51 filament

Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAbl kinase. This phosphorylation could be involved in the resistance of cancer cells to chemotherapy. We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA. The residue appeared to be on the edge of the subunit-subunit interacting site. The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal. This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation. These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site. ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak. These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure. Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament. Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA.     

Measurement of pancreatic and salivary alpha-amylase in serum: comparison of methods with five different substrates

Pancreatic and salivary alpha-amylase in human serum have been determined with the inhibitor method with 5 different substrates. An end concentration of 60 inhibitor units per liter reagent is sufficient for inhibition of the salivary alpha-amylase. The recovery of known amounts of pancreatic and salivary alpha-amylase was excellent. A mean +/- SD of 53.1 +/- 3.8% of pancreatic alpha-amylase as percentage of total alpha-amylase in serum has been observed for the 5 different substrates used. Moreover, it seems that standards obtained from crude saliva or duodenal fluid can be used as well as pure enzymes obtained from a commercial supplier.     

Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.     

Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles

We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.     

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.