STIMULATION OF ASPARTATE-AMINOTRANSFERASE FROM FARM ANIMAL SEMEN BY PYRIDOXAL 5'-PHOSPHATE

The incorporation of pyridoxal 5'-phosphate (PLP) into assay mixtures for estimating aspartate aminotransferase (AAT) activity in human semen was used in 1967. However, in the literature published since then, activity measurements of the enzyme in semen have not included the exogenous coenzyme. Our data for farm animal semen indicate that higher AAT activities are recorded when PLP is present during assay. The maximal potential catalytic activity of AAT is not achieved without exogenous PLP and the relation between the activity and the enzyme concentration can therefore be lost. We preincubated semen samples for 1 h with 0.1 mM PLP before assaying AAT activity, to check the effect of PLP. We found that the seminal phosphatases hydrolyse pyridoxal 5'-phosphate (a potential physiological substrate for these enzymes) very efficiently; EDTA was added to the preincubation mixture to maintain the coenzyme active. The activity of AAT measured with PLP in boar, ram, and bull semen was significantly higher than in assay without PLP. The highest stimulation of the enzyme by PLP was noticed for bull and ram seminal plasma (approximately 100%). FLAT activity in ram and boar semen estimated by the new procedure correlated better with sperm characteristics than the activity measured without exogenous PLP. The high amount of AAT apoenzyme in farm animal semen can be the result of easy dissociation of the coenzyme from holoenzyme. It could be caused in vitro by heating or gel filtration on Sephadex G-150 (data for ram seminal plasma). Stabilization of AAT by PLP was also observed.