Francisco Bertini

Vesicles in rat epididymal fluid. Existence of two populations differing in ultrastructure and enzymatic composition

Summary Glycosidase activity is very high in rat epididymal fluid as a consequence of the secretory capacity of the epithelium. The mechanism of this secretion is, so far, unknown. Membrane‐bound vesicles with activity of β‐galactosidase and N‐acetyl‐β‐D‐glucosaminidase were previously isolated by us from rat epididymal fluid. We report here the existence of two populations of epididymal vesicles separated by centrifugation in a sucrose gradient. They were found to differ in isopicnic equilibrium, size, ultrastructure, and enzymatic activity. Seven days after castration the protein content and specific activities of both enzymes were found decreased in the fractions containing the vesicles. A role in enzyme secretion by the epididymal epithelium is suggested for each vesicle population.

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

Summary. Fluid of rat cauda epididymidis was obtained by flushing the duct with 0.25 mol l−1 sucrose in 0.01 mol l−1 Tris‐HCl buffer pH 7.4. The fluid was centrifuged at 600 × g for 15 min and the sperm free supernatant was centrifuged at 47 000 × g for 1 h. The sediments observed with the electron microscope consisted of a heterogeneous population of membrane‐bound vesicles similar to those seen in the intact organ. In the sediment containing the vesicles the activity of β‐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‐acetyl‐galactosaminadase instead, was mainly available for the substrate and soluble in buffer containing 0.6 mol l−1 KCl. It was then inferred that β‐galactosidase is located inside vesicles with no or little affinity for the membrane, while N‐acetylglucosaminadase is bound to the external surface of vesicles. Supernatants and precipitates from suspensions of vesicles in buffered 0.5% saponin were analysed for proteins by gel electrophoresis. The electrophoretic patterns of the sediments were very different from those of supernatants and showed a number of bands greater than that of the latter. The vesicles are believed to arise from the epididymal epithelium, but their physiological role is unknown.

β-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor

In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.

Affinity sites for β‐glucuronidase on the surface of human spermatozoa

Summary. Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that β‐glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca2+‐independent, inhibited by either mannose‐6‐phosphate, phosphomannan fragments from the yeast Hansenula holstii and α‐mannosidase from the Dictyostelium discoideum, suggesting that phospho‐mannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the β‐glucuronidase binding‐sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

HIGH-AFFINITY SITES FOR beta-D-GALACTOSIDASE ON MEMBRANE-BOUND VESICLES ISOLATED FROM RAT EPIDIDYMAL FLUID

Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electronlucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1,6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.

Purification and characterization of β-galactosidase from rat epididymal fluid

Summary. β‐Galactosidase from rat epididymal fluid was purified by a combination of chromatographic techniques and precipitation with ammonium sulphate. Specific activity of the enzyme in the final precipitate was 18 times greater than in the original fluid, and it was practically free of N‐acetyl‐β‐D‐glucosaminidase. A single major band was seen when the precipitate was analysed by sodium dodecylsulphate polyacrylamide gelectrophoresis (SDS‐PAGE).

Developmental aspects of the lysosomal apparatus. Membrane affinity of N-acetyl-β-d-glucosaminidase in developing rat liver

The affinity of N-acetyl-beta-D-glucosaminidase by membranes of liver was studied in rats of different ages including fetuses at day 18 of gestation. It was found that membrane bound enzyme activity, extractable with 0.6 KCl, increases from fetal life to adulthood reaching a peak 9 days after birth. In binding assays it was found that the enzyme of fetal, 9 days old, or adult rat has high affinity for membranes of the corresponding age. These bindings were saturable and with a similar KD, but the number of receptor sites was lowest in the fetal stage, and reached a peak 9 days after birth. The fetal enzyme did not bind to adult membranes. These results suggest that the transport system of hepatic lysosomal enzymes undergoes post-natal changes which are synchronic with other parameters of lysosomal apparatus maturation studied by us and other authors as total enzyme activity and intracellular digestion of macromolecules.

Lysosomal enzyme activity in rat adrenal gland related to the postnatal development

The activity of five acid hydrolases in the adrenal gland at the perinatal stage in adult rats was measured here and changes in alpha-mannosidase and N-acetyl-beta-D- glucosaminidase activity were detected. These enzymes increase after birth reaching a peak between days 4 and 7. Other enzymes such as beta-glucuronidase, arylsulfatase and beta-glucosidase did not significantly change at the ages studied. These data suggest that the enzymatic activity and development of the adrenal gland may be correlated during the first week after birth; this is critical since most of the changes occur in this organ.

Purification of proteins from rat sperm membranes that interact with ligands other than phosphomannosyl residues.

In this study proteins were purified from rat sperm membranes which might be the high affinity sites for ligands of epididymal fluid other than the mannose‐6‐phosphate receptors. The sperm membrane proteins were solubilized and passed over an affinity column containing epididymal fluid proteins coupled to a matrix. Two bands in the range of 45–55 kDa were eluted from the column with fructose‐6‐phosphate but not with mannose‐6‐phosphate. Although the molecular weight of these proteins are similar to those of the cation‐dependent phosphomannosyl receptors they are not related. These two proteins may correspond either to two different receptors or to forms of the same receptor that recognize ligands from rat epididymal fluid. Sequencing and identification of these proteins will be the aim of future studies.