IDENTIFICATION AND IMMUNOCHEMICAL CHARACTERIZATION OF A FAMILY OF PEROXISOME MEMBRANE-PROTEINS (PMPS) IN OILSEED GLYOXYSOMES

Prior to this study the only antibodies available for characterizing p eroxisome membrane proteins (PMPs) in plants were the antibodies raise d against membranes isolated from castor bean endosperm glyoxysomes by Halpin et al. (Planta 179, 331-339 (1989)). We raised antibodies to f our different nondenatured PMP complexes solubilized in 0.63 M aminoca proate/1% dodecylmaltoside from alkaline carbonate-washed, cucumber co tyledon glyoxysome membranes. The four complexes, approximately 290/27 0, 148, 128 and 67 kDa, were excised from 5 to 10% nondenaturing gradi ent gels, passively eluted from their homogenized gel slice, concentra ted, then injected subcutaneously into rabbits. SDS-PAGE (10-15% gradi ent) of the total detergent-solubilized PMPs revealed six prominent me mbrane polypeptides: 73, 61, 52, 36, 30, and 22 kDa. The SDS-PMP compo sition of each nondenatured antigen was: PMP290/270-52, 30, 28 kDa; PM P148-30, 28, 26, 23, 22 kDa; PMP 128-73, 66, 36, 30, 23 kDa; PMP67-34, 30 kDa. These data indicated that several prominent as well as severa l minor polypeptides were common components of the PMP complexes. Thre e of the four antisera to the complexes were polyspecific, recognizing several of these common SDS polypeptides, whereas the fourth antiseru m, anti-PMP67, was monospecific for PMP30. Cross-reactivities were evi dent with each antiserum to several of these SDS PMPs from castor bean , cotton and sunflower. Affinity-purified anti-PMP30 and anti-PMP73 an tibodies specifically bound to the boundary membrane of cucumber glyox ysomes in cells examined by indirect, postembedment (LR White), immuno cytochemistry. These, and the family of other antibodies produced in t his study, provide specific molecular probes essential for elucidating biogenesis and discovering function(s) of the integral membrane prote ins in oilseed glyoxysomes.