Purification and characterization of zein-degrading proteases from germinating maize seeds

Everaldo Goncalves de Barros, Purdue University

Abstract

The molecular characterization of the machinery responsible for the degradation of storage proteins in seeds is essential for the understanding of the process of mobilization of reserves during germination. To study the degradation of zeins in maize (Zea mays L.), we have purified a set of four proteases (A$\sb1$-A$\sb4$) from germinating seeds using ammonium sulfate and isoelectric precipitations, anion exchange chromatography, and electroelution from preparative nondenaturing polyacrylamide gels. Their appearance in the endosperm of germinating seeds coincides with the onset of zein degradation. We have shown that these proteases degrade zeins dissolved in alcoholic solution as well as aggregated in protein bodies from developing maize kernels. The apparent molecular weights and net negative charges of each of these proteases are very similar. Additionally, they are inhibited by thiol-blocking agents and activated by reducing compounds. These characteristics suggest that they are a group of cysteine proteases involved in the first steps of storage protein degradation. During the purification of A$\sb1$-A$\sb4$, we detected a compound which inhibited the in vitro degradation of alpha-zeins by these proteases. This substance was purified by electroelution from a polyacrylamide gel but we did not establish if it has any physiological role or if it was artificially concentrated during enzyme purification. We attempted to isolate cDNAs encoding A$\sb1$-A$\sb4$ by screening a cDNA expression library constructed from mRNAs isolated from 3-6 DAG maize seeds, with polyclonal antibodies to these proteases. However, no immunopositives were detected. As an alternative approach, we used a cDNA (pHVEP-4) encoding a cysteine proteinase (EP-B) from the aleurone of germinating barley seeds to screen the same library. A cDNA clone (pMCP10A) encoding a putative cysteine protease of 25 kD was isolated and characterized. This enzyme showed extensive homology to the barley proteinase (71%) and also to cysteine proteases from other sources (48-63%). Genomic Southern hybridization demonstrated that the number of copies of gene(s) encoding this protease is low, 2-5 copies per haploid genome. The transcripts hybridizing with pMCP10A are only expressed during germination when A$\sb1$-A$\sb4$ are also present in the seed (2-8 DAG). Experiments to demonstrate the potential relationship between pMCP10A and these proteases are underway.

Degree

Ph.D.

Advisors

Larkins, Purdue University.

Subject Area

Molecular biology|Botany

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