Several other membrane-linked proteins were identified such as lactadherin-PAS6/7 protein, chaperone heat shock protein-71 (HSC71), vacuolar ATPase (V-ATPase), protein G-beta (G-beta) and NAP22/BASP1. One protein expressed in testis (HPAST1-testilin-EHD1) at the germ cell level was also identified. The metabolic enzymes creatine kinase (CreaKin), pyruvate kinase (PyrKin), glutamate carboxy-peptidase-like-1 (named carnosinase 1; CARN1), alpha-enolase (Enolase), aldehyde and isocitrate dehydrogenase (Ald-dh and Sorb-dh, respectively) and aldose reductase (Ald-red) were also present.
Varying amounts of lactotrans-ferin and serum albumin (OSA) were also found depending upon the preparation variable. Because these proteins were present in the caudal fluid, they may be adsorbed to the vesicle membrane. More information on these proteins is available in Table 2.
Western blots were performed using specific antibodies to confirm the identities of some of the major vesicle proteins (Fig. 5). Immunoreactions with neprilysin, ENPP3, protein G-beta, lactadherin-PAS6/7and actin were observed in raw cauda epididymal fluid (CEF) and purified vesicles (Fig. 5), but their intensity levels decreased significantly after removal of the vesicles from the caudal fluid by centrifugation (Fig. 5, CEF-hs).
FIG. 5. Immunological identification of some of the vesicle proteins. Caudal fluid (CEF), caudal fluid after high-speed centrifugation (CEF-hS), and epididymal vesicles (10 times concentrated) were separated on a 616% SDS-PAGE and transferred to nitrocellulose and probed with specific antibodies to confirm the identity of some of the proteins obtained by mass spectrometry. Similar Western blots were probed with antibodies directed against hydrophobic or lipophilic proteins known to be in the ram cauda epididymal fluid and/or on sperm membrane.