Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli

Abstract

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli. Acarbose at millimolar concentrations specifically affected the growth of E. coli K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).

FIG. 1

Structure of acarbose. The individual sugar residues are designated A to D (see the text for details).

FIG. 2

Effect of acarbose on the growth of E. coli K-12 on maltose. Cells were grown at 37°C in minimal medium (M63) in the presence of maltose (0.5%) or glucose (0.5%). At the time indicated by the arrow, acarbose was added at increasing concentrations and growth was continued for 3 h. Maltose-grown cells: ○, no addition; ●, 0.5 mM acarbose; □, 1 mM acarbose; ■, 2 mM acarbose. Glucose-grown cells: ▵, no addition; ▴ 2 mM acarbose. OD₆₅₀, optical density at 650 nm.

FIG. 3

Inhibition of [¹⁴C]maltose uptake by acarbose. Cells were grown in M63-maltose medium to the late exponential phase, harvested, washed twice in M63 salts, and resuspended to an optical density at 650 nm of 7.8. Aliquots (10 μl) were diluted in M63 salts (1 ml), and the reaction was started by adding radiolabeled maltose. At 15-s intervals, aliquots (180 μl) were withdrawn, the cells were collected by rapid filtration through OE67 membrane filters (pore size, 0.45 μm; Schleicher & Schuell), washed once with ice-cold M63 salts, and counted. Shown is a Lineweaver-Burk plot of maltose affinity recorded in the presence of different acarbose concentrations (○, 0 μM; ●, 0.5 μM; □, 2 μM; ■, 10 μM). Initial rates of transport were calculated per 10⁹ cells.

FIG. 4

Uptake of [¹⁴C]acarbose. Cells were grown and prepared for transport assays as described in legend to Fig. 3, except that the final cell suspension was adjusted to an optical density at 650 nm of 7.5. Aliquots (10 μl) were diluted in 1 ml of M63 salts, and the reactions were initiated by the addition of radiolabeled acarbose (□, ■) or radiolabeled maltose (○, ●) (final concentrations, 5.7 μM; 22 kBq). The solid symbols represent uptake of the sugars in the presence of 0.1 mM maltose and 0.1 mM acarbose, respectively. The open symbols represent transport of maltose and acarbose, respectively, in the absence of competing (unlabeled) sugars.

FIG. 5

Titration of membrane conductance induced by maltoporin with acarbose. The membrane was formed from diphytanoyl phosphatidylcholine/n-decane. Acarbose was added to the trans side of the membrane at the concentrations shown at the top of the figure. The temperature was 25°C, and the applied voltage was 20 mV.

FIG. 6

The relative conductance inhibition dependent on the acarbose concentration at one side of the membrane (trans). The data were derived from the experiment in Fig. 5. Line 1 corresponds to the fit with equation 1, assuming symmetrical binding of acarbose to maltoporin. Line 2 is the fit with equation 4. It is composed of the sum of two independent binding processes reflecting the binding from the periplasmic side (line 3; F′ = 61.3%, K′ = 12,600 M⁻¹) and from the extracellular side (line 4; F" = 38.7%, K" = 392 M⁻¹). Fits were done by least-squares analysis.

FIG. 7

Current-voltage curves of a membrane containing 890 LamB channels. The different curves were measured at acarbose concentrations ranging from 0 to 9.9 mM. The voltage is given relative to the cis side of the membrane, the side to which LamB and acarbose were added. The membrane was formed from diphytanoyl phosphatidylcholine/n-decane. The temperature was 25°C.

FIG. 8

Glucose-releasing activities of amylomaltase (MalQ) and maltodextrin glucosidase (MalZ) in the presence of maltotetraose, acarbose, or maltose. Cell extracts (80 μl) of strains HS3018 (malQ⁺ malZ⁺) (A), CB39 (malQ malZ⁺) (B), and TK38 (malQ⁺ malZ) (C), were incubated with the indicated sugars for 30 min and assayed in duplicate for the release of glucose by the GOD-POD method (5). Values represent the mean of two independent experiments. Lanes: 1, maltotetraose (10 mM); 2, acarbose (10 mM); 3, maltotetraose (10 mM) and acarbose (1 mM); 4, maltose (10 mM) and maltotetraose (1 mM); 5, maltose (10 mM), and maltotetraose (1 mM), and acarbose (1 mM); 6, maltose (10 mM) and acarbose (1 mM); 7, acarbose (10 mM) and maltose (1 mM). Standard deviations are indicated by error bars.