Identification of two distinctly localized mitochondrial creatine kinase isoenzymes in spermatozoa.
The conversion of glucose to lactate via the Embden-Meyerhof pathway yields a maximum of 2 mol of ATP per mol of glucose degraded with lesser amounts of ATP obtained if metabolic intermediates are removed for biosynthesis or if substrate cycling occurs during carbohydrate degradation. Bull sperm are an ideal test system for a quantitative estimate of ATP yield because they contain enzymes necessary for potential substrate cycling and do not carry out significant biosynthesis. Experiments utilized specifically labeled glucose and fructose to test for substrate cycling between glucose in equilibrium glucose-6-PO4, fructose in equilibrium fructose-6-PO4, and fructose-6-PO4 in equilibrium bisphosphate. Flux through the pathway (low = less than or equal to 0.05; moderate = 0.1; high = greater than 0.5 mol of carbohydrate consumed per h/10(8) cells) was altered by changing incubation temperature and/or by adding metabolic effectors. The data (in situ rates of kinases and phosphatases and metabolite crossover plots) obtained under these conditions were used to establish if the generally accepted regulatory enzymes (hexokinase and phosphofructokinase) approach the overall flux through the glycolytic pathway, i.e. that of a classical kinetically limiting rate-determining step. In summary: (a) At low flux, the rates of phosphorylation greatly exceeded overall flux. This resulted in extensive substrate cycling at all points and a net ATP yield of less than or equal to 0 mol/mol of glucose initially phosphorylated. (b) At moderate flux, rate of hexokinase approached that of overall flux through the glycolytic pathway but "excessive" phosphofructokinase activity led to substrate cycling between fructose-6-PO4 and fructose 1,6-bisphosphate and resulted in a low net ATP yield (0-0.6 mol/mol of glucose). (c) At high flux, rates of phosphofructokinase and hexokinase approached that of the overall flux. Minimal substrate cycling occurred between fructose-6-PO4 and fructose 1,6-bisphosphate, and the net ATP yield approached 1.8 mol/mol of glucose. These experiments established that the theoretical stoichiometric ATP yield of the pathway was rarely achieved during these incubations because of extensive substrate cycling. Indeed, for cauda epididymal bull sperm isolated and incubated in vitro, the glycolytic pathway serves to generate lactate, an intermediate product that is further metabolized in the mitochondria to yield the ATP that is associated with degradation of exogenous carbohydrate.