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Six chain-extended analogues of the naturally occurring glycosidase inhibitor salacinol, with ring-heteroatom variation, were synthesized for structure-activity studies with different glycosidase enzymes. The syntheses involved the reaction of PMB-protected D- and L- seleno-, thio-, and iminoarabinitol with a benzylidene- and isopropylidene-protected 1,3-cyclic sulfate, derived from commercially available D-sorbitol, in 1,1,1,3,3,3-hexafluoro-2-propanol containing potassium carbonate. Deprotection of the products afforded the novel selenonium, sulfonium, and iminium analogues of salacinol containing polyhydroxylated, monosulfated, extended acyclic chains of 6-carbons, differing in stereochemistry at the stereogenic centers and ring-heteroatom constitution. Four of these compounds inhibit recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose oligosaccharides in the small intestine, with Ki values in the micromolar range, thus providing lead candidates for the treatment of Type 2 diabetes.
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Various N-substituted derivatives of 4-O-alpha-D-glucopyranosylmoranoline have been synthesized, and their inhibitory activities against rabbit sucrase and maltase have been measured, as well as their effects on postprandial hyperglycemia in the sucrose-loaded rat, 4-O-alpha-D-Glucopyranosylmoranoline was also shown to have potent hypoglycemic activity in starch-loaded dogs.
Two beta-glucosidases (I and II) were isolated from Schizophyllum commune, and their physical and chemical properties studied. The two enzymes have very similar sequences, as shown by HPLC analysis of tryptic digests and partial amino acid sequencing. As judged by their circular dichroism spectra, they have almost identical secondary structure. The estimates for alpha-helix, beta-sheet, and other structures were 21%, 40% and 39%, respectively, for beta-glucosidase I and 27%, 32% and 41% for beta-glucosidase II. Their near-ultraviolet spectra were identical. beta-Glucosidase I was more highly glycosylated than beta-glucosidase II, having 2 mol N-acetylglucosamine/mol enzyme 36, mol mannose/mol enzyme and 1.2 mol glucose/mol enzyme vs 1.2, 17 and 3 mol/mol, respectively, in beta-glucosidase II. The native glycosylated form of beta-glucosidase I had a molecular mass of 102 kDa, and that of beta-glucosidase II, 96 kDa. As estimated from sensitivity to N-glycanase, beta-glucosidase II sugars were mainly asparagine linked, but much of the sugar in beta-glucosidase I was not removed by this treatment and was apparently serine or threonine linked. Kinetic analysis showed that both forms had similar Km values (0.3-2.1 mM) for oligosaccharides of 2-6 residues, but the kcat values of beta-glucosidase II were lower by 30-75% than those of beta-glucosidase I. The substrate dependence of kcat/Km indicated that both enzymes had binding sites for three glucose residues. The pH optimum of beta-glucosidase I was higher than that of beta-glucosidase II (5.8 vs 5.1). Both had similar specificities for several (R)-beta-D-glucosides tested. Both enzymes were competitively inhibited by their glucose product, but beta-glucosidase II was consistently less inhibited than beta-glucosidase I. Cellobiase activity was much more markedly inhibited than the activity with higher oligosaccharides, and the result of this, plus the lower hydrolytic rate with cellobiose, resulted in an accumulation of cellobiose as higher oligosaccharides were digested. Glucono-delta-lactone inhibited both enzymes and the hydrolysis of all oligosaccharide substrates similarly (Ki = 4 microM). We conclude that the catalytic site is identical in both enzymes, but subtle structural differences are reflected in a differential activity on the higher oligosaccharides and in the differential effects of the glucose product as an inhibitor. Furthermore, ethanol had a stimulatory effect on beta-glucosidase I but inhibited beta-glucosidase II, which presumably reflects differential effects of ethanol on the conformations of the two species.
1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].
(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.
The microplasmodia of Physarum polycephalum express three types of beta-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane beta-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane beta-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane beta-glucosidase 1 sequence and were highly homologous with the primary structures of fungal beta-glucosidases. Notably, the C-terminal half of membrane beta-glucosidase 1 contains two calx-beta motifs, which are known to be Ca(2+) binding domains in the Drosophila Na(+)/Ca(2+) exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane beta-glucosidase 1 differs from all previously identified family 3 beta-glucosidases. In addition to cDNA for membrane beta-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane beta-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other beta-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane beta-glucosidase is a new type family 3 enzyme fused with the Calx-beta domain. We propose that Calx-beta domain may modulate the beta-glucosidase activity in response to changes in the Ca(2+) concentration.
A digestive beta-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori beta-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori beta-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori beta-glucosidase to be a single gene. Northern blot analysis of B. mori beta-glucosidase gene confirmed larval midgut-specific expression. The B. mori beta-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori beta-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant beta-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori beta-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant beta-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per microg of recombinant B. mori beta-glucosidase. The purified recombinant B. mori beta-glucosidase showed the highest activity at 35 degrees C and pH 6.0, and were stable at 50 degrees C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori beta-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.