Rat epididymal luminal fluid acid β-d-galactosidase optimally hydrolyses glycoprotein substrate at neutral pH

Abstract

Several glycosidases, purified and characterized from mammalian tissues, have been shown to be optimally active under acidic conditions when p-nitrophenyl (PNP) or 4-methylumbelliferyl glycosides are used as substrates. Although high levels of the glycosidases are present in the epididymal lumen, their physiological role remains uncertain. To be functional, the glycosidases are expected to be enzymatically active at or near the physiological pH of luminal fluid. In this report, we demonstrate that the rat epididymal luminal fluid beta-D-galactosidase, optimally active toward PNP beta-D-galactoside at pH 3.5, shows maximum activity towards a glycoprotein substrate ([Gal-3H]fetuin) at neutral pH. Several lines of evidence, including immunoprecipitation studies using antibody to the acid beta-D-galactosidase, and substrate competition studies, indicate that PNP galactosidase and [3H]Gal galactosidase activities are caused by a single enzyme, and that the two substrates are probably cleaved by the same catalytic site(s). Competition studies with various disaccharides indicate that this enzyme is capable of cleaving a variety of galactose linkages found in both O- and N-linked oligosaccharides. Molecular-sieve column chromatography of the beta-D-galactosidase of luminal fluid under several conditions of buffer and pH show that, whereas the enzyme eluted as a tetramer (apparent M(r) 320,000) under acidic conditions (pH 3.5-4.3), only dimers and monomers (apparent M(r) 180,000 and 92,000 respectively) were observed in neutral conditions (pH 6.8). This aggregation/dissociation phenomenon is reversible. These studies indicate that beta-D-galactosidase is present in the luminal fluid in dissociated forms, and is therefore optimally active towards glycoprotein substrates at physiological pH. The potential role of the enzyme in modification of sperm surface glycoproteins is discussed.

Glycan modifying enzymes in luminal fluid of rat epididymis: Are they involved in altering sperm surface glycoproteins during maturation?

“…Two sets of enzymes, namely, glycohydrolases, which cleave sugar residues from glycoproteins and glycolipids (Conzelman and Sandhoff, 1987), and glycosyltransferases, which add sugar residues from nucleotide sugars to the acceptor site(s) on existing macromolecules (see above), are present in high concentrations in the epididymal LF that surrounds spermatozoa (Skudlarek et al, 1992;Tulsiani et al, 1993Tulsiani et al, , 1995bTulsiani et al, , 1998b. Thus, it has been suggested that glycohydrolases and glycosyltransferases may have a role in modifying sperm surface glycoproteins during epididymal maturation.…”

“…The hydrolytic enzymes are often referred to as acid hydrolases because they are optimally active at an acidic pH within the lysosome where they are known to have a degradative function. However, these enzymes are often present in extracellular fluids, such as blood (Tulsiani et al, 1977;Tulsiani and Touster, 1981), epididymal luminal fluid (Skudlarek et al, 1992;Tulsiani et al, 1995b), and female genital tract fluids (Tulsiani et al, 1996). Their degradative role (if any), when present in a neutral environment has not been elucidated.…”

Novel α‐L‐fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae)

“…The activities of a-l-fucosidases were determined using p-nitrophenyl-a-l-fucopyranoside as a substrate at the optimum pH of each enzyme. The rat epididymal fluid was purified from epididymis according to the method of Skudlarek et al [7] and used as a source of rat lysosomal glycosidases. The epididymal fluid was assayed at pH 5.2 for all lysosomal glycosidases using p-nitrophenyl glycosides as substrates.…”

A 5-Kilobase Pair Promoter Fragment of the Murine Epididymal Retinoic Acid-binding Protein Gene Drives the Tissue-specific, Cell-specific, and Androgen-regulated Expression of a Foreign Gene in the Epididymis of Transgenic Mice

“…The CAT reporter gene was preferred to the lacZ reporter gene due to the high endogenous ␤-galactosidase activity present in the epididymis (26,27), which cannot be suppressed by the usual modification of…”