Effect of swainsonine on rat epididymal glycosidases

Abstract

Summary. Each epididymis of control and swainsonine-fed rats (5 \g=m\g/mldrinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of each glycosidase. The specific-activity profile for \g=b\-glucuronidase and \g=b\-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of \g=a\-d-mannosidasein tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for \g=a\-d-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the \g=a\-d-mannosidase activity was optimal at pH 4\m=.\5and studies with highly specific antibody to lysosomal \g=a\-d-mannosidaseimmunoprecipitated all of the \g=a\-d-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.

Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions.

“…The supernatant obtained after centrifugation was assayed for PNP-mannosidase/[3H]Mang-mannosidase activities. Similar protocol has been described in our publications (36,39,41,44).…”

“…Highly specific polyclonal antibody (IgG) against rat epididymai c~-D-mannosidase was prepared as reported (44). This antibody has been shown to cross-react with the rat liver lysosomal c~-v-mannosidase (44) and rat sperm acrosomal ot-D-mannosidase (36). Anti-rat liver cytosotic c~-D-mannosidase prepared as described (35) was a gift from Dr. Oscar Toaster of this university.…”

“…1 A), which showed little or no oligosaccharide-cleaving activity. These PNP-mannosidase-rich fractions were present at the top of the gradient and likely contain the acid o~-D-mannosidase present in the luminal fluid (36) and also associated with the cytoplasmic droplets. A small amount of PNP-mannosidase activity observed in fractions 8-10 ( Fig.…”

“…The pH activity curve for the salt-washed plasma membrane fraction and for the intact sperm is shown in Fig. 5; very low activity was detected at pH 4.4, the pH optimum of the "acid" acrosomal ol-D-mannosidase (36). However, nearly 70% of the enzyme remained active at pH 7.0-7.4 (Fig.…”

“…A.eidic glycosidases, namely ~D-mannosidase, ~-D-glucuronidase, and /3-D-galaetosidase ware assayed by fluorometric method essentially as described (36). 1 U of enzyme activity is the amount of enzyme that catalyzes the release of I t~mol of 4-methylumbelliferone per hour at 37"C.…”