Metabolism of Bovine Semen. XV. Rate-limiting Reactions in the Citric Acid Cycle

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Abstract
Possible rate-limiting reactions in the citric acid cycle were investigated by measuring the evolution of C14O2 from labeled intermediates of the cycle. Ejaculated bovine spermatozoa, either washed (W), versene-treated (V), or subjected to ultrasonic vibration (S), were incubated at 37C in phosphate buffer with labeled -keto-glutarate, succinate, malate, and citrate. The C14O2 evolution was greatest with -ketoglutarate as substrate; for comparative purposes it was assigned a relative specific activity (RSA) of 100 and the yields from other substrates compared to it. With W, the RSA values were 100, 17, 16, and 11; with V, they were 100, 14, 15 and 11; and with S, they were 100, 80, 79, and 18, for -ketoglutarate, succinate, malate, and citrate, respectively. Low yields from succinate, malate, and citrate in W and V preparations are attributed to limited permeability of the cell membrane to these substrates. The S data indicate that possibly some rate limitation occurs in the succinyl coenzyme A-succinate portion of the cycle.