Figures & data
Figure 1. Formation of methylglyoxal as a by-product of the glycolytic pathway. Disturbances in the triose phosphate metabolizing enzymes TIM and GAPDH cause accumulation of dihydroxyacetone phosphate, which undergoes non-enzymatic conversion to reactive α-oxoaldehyde–methylglyoxal.
Figure 2. Inactivation of human muscle-specific enolase by methylglyoxal in 15 mM Tris-HCl buffer, pH 7.0, containing 3 mM MgSO4: black squares – modified enzyme and black circles – native enzyme. Reaction mixture containing 15 μM enolase (40 U/mg) was incubated with 2 mM MG at 37°C in the dark for 210 min. Modification process was stopped by addition of 10 μL of lysine solution (4 mg/mL) to bind the noncupled MG in reaction mixture. Aliquots containing 10 μg of protein were withdrawn at indicated time intervals for enzyme specific activity determination.
Figure 3. Inactivation of human muscle enolase by methylglyoxal in PBS. Enolase (15 μM, 40 U/mg) was incubated in PBS, pH 7.4, at 37°C in the dark for 1–180 min with different amounts of methylglyoxal: 0.54, 1.1, 2.0, 3.1, and 4.34 mM. Changes in enzyme catalytic activity are expressed in percentage of the control samples containing water instead of methylglyoxal.
Figure 4. Formation of high molecular weight derivatives of human muscle enolase by methylglyoxal glycation. Samples of 15 μM enolase after preincubaton in PBS pH 7.4, at 37°C for 30 min were modified with 4.34 mM methylglyoxal at various times (1 h, 2 h, 3 h, 26 h, 48.5 h). The reaction was stopped by addition of an excess of lysine to bind unreacted methylglyoxal. Pre-stained protein standards are marked in the left lane and unmodified enolase is in the right lane. Arrows indicate enolase advanced glycation end products and unmodified enzyme.
Figure 5. Effect of 3 mM MgSO4 and 1 mM 2-PGA on modification of human muscle enolase by methylglyoxal. (A) SDS/PAGE pattern in 10% gel: lane (1) molecular mass protein standards: fructose-6-phosphate dehydrogenase 85.2 kDa, glutamate dehydrogenase 55.6 kDa, ovalbumin 45 kDa; lane (2) native enolase; lane (3) enolase glycated in the presence of 3 mM MgSO4; lane (4) enolase modified after addition of 3 mM MgSO4 with 1 mM 2-PGA; lane (5) enolase glycated in the presence of 1 mM 2-PGA; lane (6) modification of enolase without activator ions and glycolytic substrate. Arrows indicate an enolase advanced glycation end products and unmodified enzyme. (B) Rate of inactivation of enolase with methylglyoxal in the presence: black squares – of 3 mM MgSO4, white squares – of 1 mM 2-PGA, black circles – of 3 mM MgSO4 with 1 mM 2-PGA and white circles – without activator and glycolytic substrate.
Figure 6. Susceptibility of glycation products derived from human muscle enolase to proteolytic degradation by trypsin. Electrophoretic separation of protein aggregates obtained after 19 h of reaction with MG and treated for 3 h by trypsin: lane (1) trypsin; lane (2) native enolase; lane (3) native enolase with trypsin; lane (4) enolase glycated 19 h by MG; lane (5) glycated enolase after 3 h digestion by trypsin; lane (6) molecular-mass protein standards (polymerase RNA III 165 kDa and II 155 kDa, fructose-6-phosphate kinase 85.2 kDa, BSA 68 kDa, ovalbumin 45 kDa, soybean trypsin inhibitor 21.5 kDa). Asterix indicates enolase advanced glycation end-products and unmodified enzyme.
Table I. Kinetic parameters for native and glycated human muscle enolase. Experiments were performed in conditions as described in Methods. The KM and Vmax values were determined from measurements of the initial rates of the catalytic reaction using the graphical method of Lineweaver-Burk.
Figure 7. The pH-dependence of the catalytic activity of human muscle-specific enolase modified by methylglyoxal – white circles, compared with native enzyme – black circles.
