Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa.
Glycolysis is crucial for sperm functions (motility and fertilization), but how this pathway is regulated in spermatozoa is not clear. This prompted to study the location and the regulatory properties of 6-phosphofructokinase (PFK, EC 2.7.1.11), the most important element for control of glycolytic flux. Unlike some other glycolytic enzymes, PFK showed no tight binding to sperm structures. It could readily be extracted from ejaculated boar spermatozoa by sonication and was then chromatographically purified. At physiological pH, the enzyme was allosterically inhibited by near-physiological concentrations of its co-substrate ATP, which induced co-operativity, i.e. reduced the affinity for the substrate fructose 6-phosphate. Inhibition by ATP was reinforced by citrate and H+. Above pH 8, PFK lost all its regulatory properties and showed maximum activity. However, in the physiological pH range, PFK activity was very sensitive to small changes in effectors. At near-physiological substrate concentrations, PFK activity requires activators (de-inhibitors) of which the combination of AMP and fructose 2,6-bisphosphate (F2,6P2) was most efficient as a result of synergistic effects. The kinetics of PFK suggest AMP, F2,6P2, H+, and citrate as allosteric effectors controlling PFK activity in boar spermatozoa. Using immunogold labeling, PFK was localized in the mid-piece and principal piece of the flagellum as well as in the acrosomal area at the top of the head and in the cytoplasmic droplets released from the mid-piece after ejaculation.
Kamp
G
G
Institut für Zoologie, Johannes Gutenberg-Universität, Becherweg 9-11, D-55099 Mainz, Germany.
Schmidt
H
H
Stypa
H
H
Feiden
S
S
Mahling
C
C
Wegener
G
G
D000177:Acrosome / Q000201:enzymology; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D002951:Citrates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000201:enzymology; D005635:Fructosediphosphates / Q000378:metabolism; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D010732:Phosphofructokinase-1 / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine
D002951:Citrates; D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1