PubMed · 4425409
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H Beevers, R W Breidenbach. 1974. Glyoxysomes.. https://pubmed.ncbi.nlm.nih.gov/4425409/
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The central importance of storage lipid breakdown in providing carbon and energy during seed germination has been demonstrated by isolating the genes encoding the enzymes involved in FA beta-oxidation. In contrast, little is known about the ability of germinating seeds to synthesize TAG. We report that castor cotyledons are capable of TAG synthesis. The rate of incorporation of ricinoleic acid into TAG reached a peak at 7 d after imbibition (DAI) (1.14 nmol/h/mg) and decreased rapidly thereafter, but was sustained at 20 DAI in cotyledons and true leaves. The castor DAG acyltransferase (RcDGAT) mRNA and protein were expressed throughout seed germination at levels considerably enhanced from that in the dormant seed, thus indicating new expression. Significant degradation of the RcDGAT protein was observed after 7 DAI. The DGAT activity was found to be predominantly a function of the level of the intact RcDGAT protein, with the rate of TAG synthesis decreasing as degradation of the RcDGAT protein proceeded. A possible mechanism for the degradation of the RcDGAT protein is discussed. The induction of DGAT mRNA and protein, the capacity for TAG synthesis in vitro and in tissue slices, and the differing TAG composition of dormant seed TAG vs. cotyledonary TAG provide strong circumstantial evidence for active TAG synthesis by cotyledons. However, we have not yet determined the physiological significance of this capability.
Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS). The association of a 107 kDa PEPC subunit (p107) with an immunologically unrelated bacterial PEPC-type 64 kDa polypeptide leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. COS p107 is quite susceptible to limited proteolysis during PEPC purification. An endogenous asparaginyl endopeptidase appears to catalyze the in vitro cleavage of an approximately 120 amino acid polypeptide from the N-terminal end of p107, producing a truncated 98 kDa polypeptide (p98). Immunoblotting was used to estimate proteolytic activity by following the disappearance of p107 and concomitant appearance of p98 during incubation of clarified COS extracts at 4 degrees C. The in vitro proteolysis of p107 to p98 only occurred in the combined presence of 2 mM dithiothreitol and high salt concentrations (particularly SO(4) (2-) and PO(4) (2-) salts). Although p107-degrading activity was present throughout COS development, it was most pronounced in endosperm extracts from older beans. Several protease inhibitors, including two commercially available protease inhibitor cocktails, were tested for their ability to prevent p107 proteolysis. All of the inhibitors were ineffective except for 2,2'-dipyridyl disulfide (DPDS), a relatively inexpensive and underutilized active site inhibitor of plant thiol proteases. Asparaginyl endopeptidase activity of COS extracts was unaffected by 20% (NH(4))(2)SO(4) when determined in the presence or absence of 2 mM dithiothreitol using a spectrophotometric assay based upon the hydrolysis of benzoyl-L-Asn-p-nitroanilide. Thus, we propose that the combined presence of 2 mM dithiothreitol and 20% (NH(4))(2)SO(4) promotes a p107 conformational change that exposes the N-terminal region asparaginyl residue where p107 hydrolysis is believed to occur.
Glyoxysomes are specialized peroxisomes in germinating seeds, which catalyze many reactions that convert fatty acids into carbohydrates thus generating H(2)O(2). They are characterized by the presence of catalase (CAT, E.C. 1.11.1.6) in their matrix which protects cells from oxidative stress. Here, we investigated the possibility that a protein can be protected from oxidative damage by its association with CAT. We purified peroxisomal CAT from germinating castor beans by ion exchange, gel filtration, and hydroxylapatite chromatography. Gel filtration of the matrix proteins, cross-linking, and co-immunoprecipitation studies indicate that CAT associates with a glyoxysomal matrix protein, isocitrate lyase (ICL, E.C. 4.1.3.1). In addition, we found that H(2)O(2) inactivates ICL and degrades its product, glyoxylate, when CAT is inactive. ICL and its product appear to be sensitive to oxidative damage; thus, association of CAT with ICL would afford protection from H(2)O(2).