Purification and Characterization of Zein-Degrading Proteases from Endosperm of Germinating Maize Seeds

Barros, Edvaldo; Larkins, Brian A.

doi:10.1104/pp.94.1.297

Cited by 60 publications

(21 citation statements)

References 22 publications

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“…Attempts to minimize protein degradation by adding protease inhibitors to membrane preparations obviously will not control the activities of these enzymes. Other membrane-bound proteases that were identified in plant cells (5,8,20) have properties quite distinct from the Chlamydomonas enzyme.…”

Section: Membrane Protease From Chlamydomonasmentioning

confidence: 99%

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

Hoober¹,

Hughes²

1992

Plant Physiol.

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In Chiamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-1 13, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.

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Section: Membrane Protease From Chlamydomonasmentioning

confidence: 99%

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

Hoober¹,

Hughes²

1992

Plant Physiol.

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“…This indicates that the inability to germinate is a consequence of a nutritional deficiency. Since y-zein is the first storage protein hydrolyzed during germination (22,23), this suggests that the amino acids provided by this protein are essential for early development of the seedling.…”

Section: Discussionmentioning

confidence: 99%

opaque-15, a maize mutation with properties of a defective opaque-2 modifier.

Dannenhoffer

Bostwick

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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An opaque mutation was identified that reduces 'y-zein synthesis in maize endosperm. The mutation, opaque-15, causes a 2-to 3-fold reduction in y-zein mRNA and protein synthesis and reduces the proportion of the 27-kDa y-zein A gene transcript. Although the protein bodies in opaque-15 are similar in size and morphology compared to wild type, there are fewer of them in developing endosperm cells. The opaque-15 mutation maps near the telomere of chromosome 7L, coincident with an opaque-2 modifier locus. Based on its phenotype, opaque-15 appears to be a mutation of an opaque-2 modifier gene.Mutations that reduce the synthesis of maize seed storage proteins, zeins, typically result in endosperm with a soft, starchy phenotype. Some of these mutations, notably opaque-2 (o2) and floury-2 (fl2), increase the synthesis of other endosperm proteins (1, 2), resulting in a higher lysine content (3,4). Many years of effort were devoted to introgressing o2 into agronomically important maize genotypes to create high-lysine corn. However, the soft, starchy endosperm of the mutant kernel caused it to be susceptible to mechanical damage and insect pests and ultimately prevented wide utilization of o2 (5).In recent years, the introgression of genes known as o2 modifiers has solved many of the problems associated with the soft phenotype of o2 mutants (6). The modifiers increase seed density, vitreousness, and y-zein content of o2 mutants (7). Activity of the modifier genes is associated with a 2-to 3-fold increase in the synthesis of the 27-kDa y-zein mRNA and protein (8, 9) and appears to involve posttranscriptional regulation of y-zein gene expression (9). Several breeding programs have used the modifier genes to develop vitreous genotypes with a high lysine content that are referred to as Quality Protein Maize (6). Although o2 modification behaves as a complex quantitative trait (10), recent genetic evidence suggests that as few as two genes may be involved (11). In addition, two loci involved in o2 modification have been identified by restriction fragment length polymorphism (RFLP) mapping (11); one locus maps near the centromere of chromosome 7, and the second maps near the telomere on the long arm of chromosome 7.To isolate o2 modifier genes and determine their mode of action, we screened a group of mutant seeds for reduced levels of the 27-kDa y-zein protein. Here we report an opaque mutation, designated opaque-15 (o15), that significantly reduces y-zein mRNA and protein without significantly affecting the accumulation of other zeins. The mutation maps near an o2 modifier and, like an o2 modifier, alters the ratio of mRNAs encoded by the A and B y-zein genes. Understanding the biochemical basis of olS will help clarify the regulation of ,y-zein gene expression and the factors responsible for development of vitreous endosperm in maize. Although the terms vitreous and opaque describe mature kernel phenotypes, developing kernels that are wild type (i.e., 015) will be called vitreous, whereas mutant kernels (i.e., olS) wi...

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“…These data indicate that the blockage of protein degradation caused by ABA was due to the inhibition of protease activity. In corn endosperm, the onset of storage protein degradation is attributed to initial protease activity (Barros & Larkins, 1990).…”

Section: Aba Effect On the Protein Mobilization And Protease Activitymentioning

confidence: 99%

“…The protocol of electrophoresis in Activity Gels, obtained for proteases in A. peregrina cotyledons, was based on Asahi et al (1985) and Barros and Larkins (1990).…”

Section: Electrophoresismentioning

confidence: 99%

“…Qualitative studies on the effects of these growth regulators during germination have used the electrophoresis and its derivations as the main techniques to observe the pattern of protein and mRNAs synthesis induced by these regulators (Paiva & Kriz, 1994;Barros & Larkins, 1990;Borges et al, 1990;Nolan & Ho, 1988;Hammerton & Ho, 1986;Asahi et al, 1985). In this work, the denaturating SDS-PAGE and nondenaturating PAGE -Activity Gels (polyacrylamide gels copolymerized with substrate for enzymes) systems were used in order to observe the polypeptide profile and protease activity, in embryos as well as in cotyledons of A. peregrina.…”

Section: Introductionmentioning

confidence: 99%

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Effect of ABA and GA3 on protein mobilization in embryos and cotyledons of angico [Anadenanthera peregrina (L.) speg] seeds during germination

Barduche

Paiva

Lopes

et al. 1999

Braz. arch. biol. technol.

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Purification and Characterization of Zein-Degrading Proteases from Endosperm of Germinating Maize Seeds

Cited by 60 publications

References 22 publications

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

opaque-15, a maize mutation with properties of a defective opaque-2 modifier.

Effect of ABA and GA3 on protein mobilization in embryos and cotyledons of angico [Anadenanthera peregrina (L.) speg] seeds during germination

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