VMAX OF MITOCHONDRIAL ELECTRON SHUTTLES IN RAT SKELETAL MUSCLE AND LIVER

Abstract

Aerobic glycolytic flux demands a commensurate net transport of reducing equivalents (e-) into mitochondria (MITO). However, because the MITO matrix redox potential is maintained more negative than the cytosol, net e-shuttling requires the application of an energetic driving force. Two well known e- shuttles, the malate-aspartate (MA) and glycerol-3-phosphate (G3P) shuttles, are driven either by delta psi (MA) or via linkage to a more positive FAD-linked redox center (G3P). Recently lactate has been proposed as an additional molecular carrier of e- into MITO. We evaluated the catalytic potentials of these three shuttling systems in MITO isolated from soleus (type I) and superficial vastus (type IIb) rat skeletal muscle and also liver (n = 5 MITO preps per tissue). MITO were incubated in KCl-based media at 37C while O2 consumption (Jo) was measured polarographically. In the presence of saturating ADP, MA shuttle Vmax was assessed using 10 mM glutamate + 1 mM malate + 1 mM arsenite, the G3P shuttle using 10 mM G3P, and the lactate shuttle as 10 mM lactate + 1 mM malate. Pyruvate 1 mM + malate 1 mM (P+M) was also assessed. Data are presented in units of nmol O2/min/mg mito protein as means + SE, and are listed in the following order for each tissue: P+M; MA Shuttle; G3P Shuttle; Lactate Shuttle. Type I: 712+8; 411+51; 11.0+1.3; 9.6+5.2. Type IIb: 639+69; 276+30; 259+9; 4.2+1.8. Liver: 35.2+4.5; 37.1+7.8; 2.5+0.6; 4.0+1.4. Type IIb MITO demonstrated high Jo for both the MA and G3P shuttles, and these rates were fully additive when both shuttles were active (not shown). Lactate gave Jo of 1.5% and 1.6% the MA and G3P shuttles, respectively. Type I and liver MITO had low Jo with G3P, but high MA shuttle Vmax, as previously shown. Laclate Jo was 2.3% and 10.8% of MA shuttle Jo in type I and liver, respectively. We conclude that: 1) the results are consistent with energetically-driven redox separation of the cytosol from the matrix and 2) MITO in fast glycolytic muscle are robustly equipped to shuttle e- from the cytosol. Supported by NSF #IBN-0116997.