Chemie und Biochemie mikrobieller α‐Glucosidasen‐Inhibitoren

Truscheit, Ernst; Frommer, W.; Junge, Bodo; Müller, Lutz; Schmidt, Delf; Wingender, W.

doi:10.1002/ange.19810930905

Cited by 151 publications

(33 citation statements)

References 66 publications

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“…A batch ofaplanin (BAY e 4609), containing 20% pseudooligosaccharides of DP 9-14 of the acarbose type (10,36) and 80% non-inhibitory oligosaccharides, was kindly donated by Drs. E. TRUSCHEIT and D. SCHMIDT, Bayer AG, Wuppertal, F.R.G.…”

Section: Methodsmentioning

confidence: 99%

Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley α-amylase 2

Gibson

Svensson

1987

Carlsberg Res. Commun.

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In barley a-amylase 2, two and three tryptophans are protected against reaction with dimcthyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide by 13-cyclodextrin and the pseudooligosaccharide inhibitor aplanin, respectively.Fragments were generated from the enzyme derivatives by digestion with Armillaria mellea protease and trypsin, and isolated by RP-HPLC. The substituted tryptophans were identified by amino acid and sequence analyses of modified peptides. Aplanin and 13-cyclodextrin both reduced the accessibility of Trp276 and -277. In addition, aplanin hindered modification of Trp206, and only this derivative retained activity. Trp206 probably belongs to the active site region, whereas Trp276 and -277 are located in a different binding site. This suggestion is supported by a comparison with the 3-D structure of Taka-amylase A guided by sequence homology between it and barley a-amylase.

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Section: Methodsmentioning

confidence: 99%

Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley α-amylase 2

Gibson

Svensson

1987

Carlsberg Res. Commun.

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“…Effect of ligands during carbodiimide treatment The accessibility of essential carboxyl groups in glucoamylase was tested by reacting the enzyme with a fixed concentration of EDC and glycine ethyl ester at pH 4.7 in the presence of various specific ligands. The ki,=t values (Table I) reflected the tight-binding inhibitor acarbose (57) to be superior to the substrate maltose as protective agent while both a transition state analoguegluconolactone and a competitive inhibitor maltitol (21, 23) provided marginal protection. The effect of acarbose was concentration-dependent (Fig.…”

Section: Inactivation Of Glucoamylase With Carbodiiinidesmentioning

confidence: 99%

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Svensson

Møller

Clarke

1988

Carlsberg Res. Commun.

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Glucoamylases GI and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC). A tolal of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the GI and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pK~ 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these fig,ands, differenlial labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC.Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of K~ and a slight reduction of V,~ for hydrolysis of starch relative to the unmodified enzyme. Both K~ and Vm~ for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC.In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

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“…Recent work in our laboratory had demonstrated that compounds like conduritol-B-epoxide and Triton X-I00 P/J [17], proved to be similarly ineffective. In contrast, nojirimycin and 1 -deoxynojirimycin were rather potent inhibitors for both trimming glucosidase I and 11. and Nph-x-glucosides ( x --~ x j as substrate Nojirimycin and 1 -deoxynojirimycin are basic glucose analogues, dNM dirrering from nojirimycin by the lack of the anomeric hydroxyl group.…”

Section: Inhibition Studiesmentioning

confidence: 99%

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine‐linked oligosaccharides

Hettkamp

Legler

Bause

1984

European Journal of Biochemistry

168

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Trimming glucosidase 1 and I1 have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and I1 and the nonspecific cc-glucosidase(s) were inhibited by 1 -deoxynojirimycin with median inhibitory concentration of 3 pM, 20 pM, 12 pM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of I-deoxynojirimycin and formed the basis for the design of the affinity ligand.Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecullar mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was % 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 -'C in the presence of detergent (t,,, z 20 days) and showed high specificity for the hydrolysis of the terminal ( E 1,2)-linked glucose residue in the natural substrate Glc,-Man,-(GlcNAc),.During N-glycoprotein formation oligosaccharides of the composition Glc,-Man,-(GlcNAc), are preassembled on a lipid carrier (dolichyl diphosphate, Doil-PP) and subsequently transferred 'en bloc' onto target asprragine residues of the nascent polypeptide chain. The protein-bound oligosaccharides are then modified by a sequence of reactions, generating N-glycans with either high mannoge orland complex type structures. The trimming sequence is initiated by the stepwise removal ofthe three glucose units, which is catalysed by at least two specific glucosidases (glucosidase I and 11). Cleavage of the glucose residues is followed by an einzymatic hydrolysis of several ( a 1,2)-linked mannose residues, resulting in high mannosc structures, which may be preserved as such or further modified to complex type oligosaccharides, the latter process requiring the concerted action of presumably other rx-mannosidases and various glycosyltransferhses (for review, see [I]).Since glucose residues are generally not found in mature N-glycoproteins, it is suggested that their transient occurrence may have an important function in the regulation of oligosaccharide trimming and processing. First evidence that the presence of the Glc, unit may give a signal for the transfer of the lipid-linked precursor oligosaccharide to protein, came from studies ofTurco et al. [2], who demonstrated that, at least under conditions in vitvo, the Glc,-containing structure is transferred more rapidly to protein acceptors than the intermediate devoid of glucose. A similar regulatory function was recently put forward by Spiro et al. [3], who discussed that the extent of N-glycosylation might depend...

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Chemie und Biochemie mikrobieller α‐Glucosidasen‐Inhibitoren

Cited by 151 publications

References 66 publications

Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley α-amylase 2

Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley α-amylase 2

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine‐linked oligosaccharides

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