Alteration of the isoform composition of plasma-membrane-associated rat sperm alpha-L-fucosidase during late epididymal maturation: comparative characterization of the acidic and neutral isoforms.
In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum alpha-L-fucosidase in rat sperm. In the present study, rat sperm alpha-L-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of alpha-L-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53+/-2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of alpha-L-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. alpha-L-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and alpha-L-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH-activity curves (with optima near pH 7) and comparable apparent KM values (0.4+/-0.04 mM) for 4-methylumbelliferyl alpha-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver alpha-L-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm alpha-L-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.
Abascal
I
I
Section of Histology and General Embryology, Department of Cell Biology, Medical School, University of Murcia, Murcia, Spain E-30071, USA.
Skalaban
S R
SR
Grimm
K M
KM
Avilés
M
M
Martianez-Menarguez
J A
JA
Castells
M T
MT
Ballesta
J
J
Alhadeff
J A
JA
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004822:Epididymis / Q000033:anatomy & histology / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D013079:Sperm Maturation; D013094:Spermatozoa / Q000201:enzymology*; D013696:Temperature; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D005644:alpha-L-Fucosidase