First observations on enzymatic activity and protein content of vesicles separated from rat epididymal fluid.

FORNES MW; BARBIERI A; SOSA MA; BERTINI F

ANDROLOGIA

WILEY-BLACKWELL PUBLISHING, INC

Año: 1991 vol. 23 p. 347 - 351

0303-4569

Fluid of rat cauda epididymidis was obtained by flushing the duct with 0.25 mol l-1 sucrose in0.01 mol l-1 Tris-HCl buffer pH 7.4. The fluid was centrifuged at 600 x g for 15 min and thesperm free supernatant was centrifuged at 47,000 x g for 1 h. The sediments observed with theelectron microscope consisted of a heterogeneous population of membrane-bound vesiclessimilar to those seen in the intact organ. In the sediment containing the vesicles the activity ofbeta-galactosidase was mostly unavailable for the substrate showing a high degree of latency:the activity became soluble after a treatment with 0.5% saponin. The activity of N-acetylgalactosaminadaseinstead, was mainly available for the substrate and soluble in buffercontaining 0.6 mol l-1 KCl. It was then inferred that beta-galactosidase is located inside vesicleswith no or little affinity for the membrane, while N-acetylglucosaminadase is bound to theexternal surface of vesicles. Supernatants and precipitates from suspensions of vesicles inbuffered 0.5% saponin were analysed for proteins by gel electrophoresis. The electrophoreticpatterns of the sediments were very different from those of supernatants and showed a numberof bands greater than that of the latter. The vesicles are believed to arise from the epididymalepithelium, but their physiological role is unknown.