Biology of Reproduction, Vol 27, 1239-1246, Copyright © 1982 by Society for the Study of Reproduction

ARTICLES

Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa

PY Wong and AY Tsang

A glycoprotein of molecular weight 32K has been isolated and purified from the rat caudal epididymal fluid by gel filtration, ion-exchange and affinity chromatography. The highly purified protein was labeled with radioactive iodine and the binding of the 125I-labeled 32K rat epididymal protein (REP) to washed rat caudal epididymal sperm was studied under various conditions. Scatchard plots of the binding data revealed two binding kinetics. One bound with high affinity (KD = 2.6 X 10(-10) ) but low capacity. The other bound with lower affinity (KD = 2.2 X 10(-9)M) but high capacity. The rate of binding of the labeled protein to sperm was dependent on the temperature of the incubation medium. At the scrotal temperature of 33 degrees C, maximal binding was obtained after 40 min. However, at 22 degrees C equilibrium state was reached after 90 min and at 0 degrees C, the equilibrium rate was not reached even after 120 min of incubation. Binding showed dependence on extracellular pH (optimal pH at 4) and ionic strength of the incubation medium. High ionic strength was found to inhibit binding of the 125I- labeled 32K REP to rat caudal epididymal sperm. Specific binding was abolished by 100-fold molar excess unlabeled 32K REP or by native rat caudal epididymal fluid proteins, but not by albumin or ovalbumin. This indicates high specificity of binding. This study has provided direct evidence for the interaction of an epididymal protein with epididymal spermatozoa.

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