Ascorbate peroxidase. A prominent membrane protein in oilseed glyoxysomes.

The glyoxysomes of growing oilseed seedlings produce H2O2, a reactive oxygen species, during the beta-oxidation of lipids stored in the cotyledons. An expression library of dark-grown cotton (Gossypium hirsutm L.) cotyledons was screened with antibodies that recognized a 31-kD glyoxysomal membrane polypeptide. A full-length cDNA clone (1258 bp) was isolated that encodes a 32-kD subunit of ascorbate peroxidase (APX) with a single, putative membrane-spanning region near the C-terminal end of the polypeptide. Internal amino acid sequence analysis of the cotton 31-kD polypeptide verified that this clone encoded this protein. This enzyme, designated gmAPX, was immunocytochemically and enzymatically localized to the glyoxysomal membrane in cotton cotyledons. The activity of monodehydroascorbate reductase, a protein that reduces monodehydroascorbate to ascorbate with NADH, also was detected in these membranes. The co-localization of gmAPX and monodehydroascorbate reductase within the glyoxysomal membrane likely reflects an essential pathway for scavenging reactive oxygen species and also provides a mechanism to regenerate NAD+ for the continued operation of the glyoxylate cycle and beta-oxidation of fatty acids. Immunological cross-reactivity of 30- to 32-kD proteins in glyoxysomal membranes of cucumber, sunflower, castor bean, and cotton indicate that gmAPX is common among oilseed species.

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 peroxisome membrane proteins (PMPs) in plants were the antibodies raised against membranes isolated from castor bean endosperm glyoxysomes by Halpin et al. (Planta 179, 331-339 (1989)). We raised antibodies to four different nondenatured PMP complexes solubilized in 0.63 M aminocaproate/1% dodecylmaltoside from alkaline carbonate-washed, cucumber cotyledon glyoxysome membranes. The four complexes, approximately 290/270, 148, 128 and 67 kDa, were excised from 5 to 10% nondenaturing gradient gels, passively eluted from their homogenized gel slice, concentrated, then injected subcutaneously into rabbits. SDS-PAGE (10-15% gradient) of the total detergent-solubilized PMPs revealed six prominent membrane polypeptides: 73, 61, 52, 36, 30, and 22 kDa. The SDS-PMP composition of each nondenatured antigen was: PMP290/270-52, 30, 28 kDa; PMP148-30, 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 several minor polypeptides were common components of the PMP complexes. Three of the four antisera to the complexes were polyspecific, recognizing several of these common SDS polypeptides, whereas the fourth antiserum, anti-PMP67, was monospecific for PMP30. Cross-reactivities were evident with each antiserum to several of these SDS PMPs from castor bean, cotton and sunflower. Affinity-purified anti-PMP30 and anti-PMP73 antibodies specifically bound to the boundary membrane of cucumber glyoxysomes in cells examined by indirect, postembedment (LR White), immunocytochemistry. These, and the family of other antibodies produced in this study, provide specific molecular probes essential for elucidating biogenesis and discovering function(s) of the integral membrane proteins in oilseed glyoxysomes.

Identification of Peroxisome Membrane Proteins (PMPs) in Sunflower (Helianthus annuus L.) Cotyledons and Influence of Light on the PMP Developmental Pattern.

Boundary membranes were recovered from glyoxysomes, transition peroxisomes, and leaf-type peroxisomes purified from cotyledons of sunflower (Helianthus annuus L.) at three stages of postgerminative growth. After membranes were washed in 100 mM Na2CO3 (pH 11.5), integral peroxisome membrane proteins (PMPs) were solubilized in buffered aminocaproic acid/dodecyl maltoside (0.63 M/1.5%) and analyzed by nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Six prominent nondenatured PMP complexes and 10 prominent SDS-denatured polypeptides were identified in the membranes of the three types of peroxisomes. A nondenatured complex of approximately 140 kD, composed mainly of 24.5-kD polypeptides, decreased temporally, independently of seedling exposure to white, blue, or red light; only far-red light seemed to prevent its decrease. PMP complexes of approximately 120 and 70 kD, in contrast, were present at all stages and changed in polypeptide content. It remains to be determined whether these data reflect changes within in vivo complexes or within complexes formed following/during detergent solubilization. Conversion of glyoxysomes to leaf-type peroxisomes in white or red light after a 2-d dark period was accompanied by the appearance of three SDS-denatured PMPs: 27.5, 28, and 47 kD. The former two became part of the PMP120 and 70 complexes, as well as part of a new PMP130 complex that also possessed the PMP47. Growth of seedlings in blue or far-red light did not promote the appearance of PMPs 27.5 or 28. Blue light promoted the appearance of PMP47, and far-red light seemed to prevent its appearance. Chlorophyll likely is not the photoreceptor involved in accumulation of PMPs because the PMP composition is distinctly different in seedlings irradiated with red or blue light of comparable fluence rates. Several lines of evidence indicate that the synthesis and acquisition of membrane and all matrix proteins are not coupled. The data provide evidence for a change in PMP composition when sunflower or any other oilseed glyoxysomes are converted to leaf-type peroxisomes and suggest that the change is regulated by both photobiological and temporal mechanisms.