A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside

Abstract

A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.

Keywords: Drosophila melanogaster mannosidase II; GMII; Golgi mannosidase II; Res-Man; continuous fluorescent enzyme assay; dGMII; fluorogenic substrate; kinetic assay; resorufin α-D-mannopyranoside.

Figure 1

Res-Man Synthetic Scheme.

Figure 2

Continuous fluorescent analysis of recombinant golgi α-mannosidase II (dGMII) from Drosophila melanogaster (△) and recombinant human lysosomal mannosidase (HLM) (◊) using the substrate resorufin-α-D-mannopyranoside (Res-Man). Res-Man (500μM, diluted from 5mM DMSO stock using 100 mM sodium acetate buffer, pH 6.0) (50μL) was added to the enzyme samples (50 μL) to a final concentration of 250 μM. Enzyme samples were prepared in 100 mM sodium acetate buffer and diluted to a final concentration of (6.67 μg/mL) in the reaction mixture. Fluorescence of a blank (no-enzyme) sample (□) was also analyzed over the same time period. Fluorescence emission was measured at 590 nm with excitation at 550 nm for 40 min at 1-min intervals. Data points are means, and error bars represent standard errors (n = 3).

Figure 3

(a.) Inhibition of recombinant golgi α-mannosidase II (dGMII) from Drosophila melanogaster by swainsonine (SIGMA 068K8721), using the substrate resorufin- α -D-mannopyranoside (Res-Man). IC₅₀ was determined to be 100nM and K_i was determined to be 44.7nM. DGMII was diluted to 6.67 μg/mL in 100mM Sodium Acetate, pH 6.0. Swainsonine was added at concentrations of 10μM (●), 1μM (▼), 500nM (▽), 250nM (▲), 100nM (△), 50nM (■), 25nM (□), and 10nM (◊). An enzyme sample containing no inhibitor (○) was also prepared. Res-Man (500μM, diluted from 5mM DMSO stock using sodium acetate buffer) (50μL) was added to the enzyme samples (50μL) to a final concentration of 250 μM. Fluorescence of all assays was recorded using EX: 550; EM: 595. Data were converted to μmol of resorufin product produced using a standard curve created by measuring fluorescence of several known concentrations of resorufin in sodium acetate buffer (pH 6.0). Data points are means, and error bars represent standard errors (n = 3). (b.) Lineweaver-Burk plots generated from kinetic assay using the substrate resorufin-α-D-mannopyranoside (Res-Man). The kinetic parameters K_m and V_max were determined via computer-assisted nonlinear regression analysis using GraFit (Erithacus Software). Values for K_m and V_max for the uninhibited assay were determined by GraFit to be 202.05 μM and 0.00059442 μmol/ml/min, respectively. Inset shows competitive inhibition by swainsonine at 50 nM (△) and 250 nM (□) concentrations.

Figure 3

(a.) Inhibition of recombinant golgi α-mannosidase II (dGMII) from Drosophila melanogaster by swainsonine (SIGMA 068K8721), using the substrate resorufin- α -D-mannopyranoside (Res-Man). IC₅₀ was determined to be 100nM and K_i was determined to be 44.7nM. DGMII was diluted to 6.67 μg/mL in 100mM Sodium Acetate, pH 6.0. Swainsonine was added at concentrations of 10μM (●), 1μM (▼), 500nM (▽), 250nM (▲), 100nM (△), 50nM (■), 25nM (□), and 10nM (◊). An enzyme sample containing no inhibitor (○) was also prepared. Res-Man (500μM, diluted from 5mM DMSO stock using sodium acetate buffer) (50μL) was added to the enzyme samples (50μL) to a final concentration of 250 μM. Fluorescence of all assays was recorded using EX: 550; EM: 595. Data were converted to μmol of resorufin product produced using a standard curve created by measuring fluorescence of several known concentrations of resorufin in sodium acetate buffer (pH 6.0). Data points are means, and error bars represent standard errors (n = 3). (b.) Lineweaver-Burk plots generated from kinetic assay using the substrate resorufin-α-D-mannopyranoside (Res-Man). The kinetic parameters K_m and V_max were determined via computer-assisted nonlinear regression analysis using GraFit (Erithacus Software). Values for K_m and V_max for the uninhibited assay were determined by GraFit to be 202.05 μM and 0.00059442 μmol/ml/min, respectively. Inset shows competitive inhibition by swainsonine at 50 nM (△) and 250 nM (□) concentrations.

Figure 4

Computer-assisted nonlinear regression kinetic analysis of resorufin-α-D-mannopyranoside (Res-Man). Assay method was performed by adding recombinant golgi α-mannosidase II (dGMII) from Drosophila melanogaster (6.67 μg/mL final conc.) to four concentrations (0.50, 0.30, 0.20, 0.10 mM) of Res-Man in 100 mM sodium acetate buffer, pH 6.0. Fluorescence was recorded (EX: 550, EM: 595) at 30-s time intervals for 210 s. Data were converted to μmol of resorufin product produced using a standard curve created using known concentrations of resorufin in sodium acetate buffer (pH 6.0). Data points are means, and error bars represent standard errors (n = 3).