Archive for the ‘Plasma’ Category - Part 2

Secreted Epididymal Glycoprotein: RESULTS(10)

A synthetic degenerate oligonucleotide probe based on the major peptide sequence was used to screen a filter-based cDNA library prepared from whole rat epididymis in p-SPORT1 vector. Twenty-seven independent clones were selected that hybridized to the oligonucleotide probe. A single clone containing a plasmid with the largest insert was chosen for further analysis. The insert […]

Secreted Epididymal Glycoprotein: RESULTS(9)

Sequence Analysis of the 24-kDa Antigen from Cauda Spermatozoa To obtain clues to the functionality of the 24-kDa form of 2D6 antigen, it was subjected to a primary sequence analysis followed by database searches to identify potential orthologues in other tissues. For this purpose, the reduced 24-kDa subunit was excised from nonreducing/reducing two-dimensional PAGE gels […]

Diethylstilbestrol Versus Estradiol: DISCUSSION(8)

In conclusion, both the early and long-term development of the cervix in intact hamsters was differentially disrupted by neonatal exposure to the same high dose of DES versus E2. The extent of this phenomenon in adult animals may be caused, at least in part, by differing degrees of altered ovarian function between the two neonatal […]

Secreted Epididymal Glycoprotein: RESULTS(8)

The continued presence of the 24-kDa antigen on blots after alkaline hydrolysis was demonstrated using a polyclonal antibody prepared to the 24-kDa antigen excised from the gel (Fig. 9). As an additional control, parallel blots were probed with 2B1 McAb, which has been shown to recognize a peptide epitope. Alkali treatment had no detectable effect […]

Secreted Epididymal Glycoprotein: RESULTS(7)

When parallel blots were probed with a polyclonal antibody to rat CD52, a positive reaction was obtained in the supernatant from PI-PLC treated sperm. Preextraction of cauda spermatozoa with 1 M KCl to remove loosely bound glycoproteins from the plasma membrane did not make 2D6 antigen susceptible to cleavage by PI-PLC (results not shown).

Secreted Epididymal Glycoprotein: RESULTS(6)

Two methods widely used for assessing protein-lipid interactions are partitioning of the antigen into Triton X-114 and its solubilization by PI-specific PLC (PI-PLC). The latter procedure indicates the presence of a GPI anchor, although not all GPI-anchored proteins on cell surfaces are susceptible to PI-PLC cleavage owing to problems of steric hindrance. Following solubilization and […]

Secreted Epididymal Glycoprotein: RESULTS(5)

Binding of2D6 Antigen to the Sperm Plasma Membrane Secreted epididymal glycoproteins are known to bind to spermatozoa with varying tenacities. To elucidate the strength and nature of the binding of the 24-kDa form of 2D6 antigen to the sperm plasma membrane, cauda epidi-dymidal spermatozoa were washed in PBS followed by resuspension in different dissociating agents, […]

Secreted Epididymal Glycoprotein: RESULTS(4)

The pronounced effects of disulfide bond cleavage on the size of the proteins recognized by 2D6 McAb suggests that they may be secreted as a multimeric complex and are progressively ‘‘processed’’ by reduction and endoproteo-lysis as they pass through the distal regions of the duct. Further evidence to support this interpretation is shown by Western […]

Secreted Epididymal Glycoprotein: RESULTS(3)

Identification of Secreted and Membrane-Bound Forms of 2D6 Antigens in Luminal Plasma and Spermatozoa from the Caput and Cauda Epididymidis Proteins in epididymal plasma and detergent extracts of spermatozoa collected by micropuncture were analyzed under nonreducing and reducing conditions (Fig. 3). Under nonreducing conditions, 2D6 McAb recognized a high-molecular-mass protein of ~250 kDa in caput […]

Secreted Epididymal Glycoprotein: RESULTS(2)

Large empty vesicles adjacent to the Golgi were generally unlabeled. The surfaces of microvilli protruding from the apical surface of principal cells were encrusted with gold particles that were also present in the duct lumen, where they had a random distribution (Fig. 1F). Membrane-bound vesicles could not be positively identified in the lumen and gold […]

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