DETECTION AND IMMUNOLOCALIZATION OF GLYCOPROTEINS OF THE PLASMA-MEMBRANE OF MAIZE SPERM CELLS

After purifying plasma membranes from isolated maize sperm cells by aq ueous polymer two-phase partition, peripheral and integral proteins we re solubilized from the plasma membrane with Triton X-114 and separate d in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA GE). Silver staining revealed 10 bands (19-68 kDa) of peripheral membr ane proteins and about 40 bands (12-120 kDa) of integral proteins. Per oxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51, 48, 44, 36, and 32 kDa, and 6 integr al polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands diff ered from those of somatic samples. Staining specificity was demonstra ted by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peri pheral 68 kDa polypeptide was further separated into 4 spots by isoele ctric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D ) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membra ne of maize sperm cells via the fluorescein isothiocyanate (FITC) tech nique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous lab elling of the surface of isolated viable maize sperm cells with Con A- FITC.