Figure 4
(A,B) The activity of rabbit muscle LDH and porcine heart LDH was measured in the presence of a large excess of rabbit muscle GAPDH. (A) steady-state activities of rmLDH (10 nM) or phLDH (17 nM) were measured at fixed NADH concentration (40 µM) in the presence of increasing concentration of rmGAPDH (100 to 240 μM of NADH binding sites). Increase in rmGAPDH concentration leads to the disproportional decrease in the measured and the calculated free-diffusion activities (lower panels) which results in the increase in the ratio between the two activities (upper panels, and Eq. 2−3). These results indicate that LDH molecules can use GAPDH-NADH complex as a substrate in addition to free NADH, i.e. NADH channeling (Supp. Fig. 11). (B) steady-state activities of rmLDH (10 nM) or phLDH (17 nM) were measured in the presence of decreasing NADH concentration with rmGAPDH fixed at 200 μM (NADH binding sites). The decrease in NADH concentration leads to the disproportional decrease in the measured and the calculated free-diffusion activities (lower panels), which results in increase in the ratio between the two activities (upper panels, and Eq. 2-3). The red curve represents the Michaelis-Menten profile for LDH activity with the rmGAPDH-NADH complex as the substrate, which was calculated by subtracting the calculated free-diffusion profile from the measured profiles (Supp. Fig. 14). The calculated apparent KM constant for rmLDH is 35 ± 5.5 μM and 59 ± 6 μM for phLDH (Table 3). Thus, the observed KM constants are a result of competition between the channeled and free-diffusion paths (Supp. Fig. 11).
