Effect of γ-radiation on the plasma and vacuolar membranes of cultured spinach cells

Mitsuhashi, Naoto; Koshiba, Tomokazu; Sato, Momoko

doi:10.1016/s0031-9422(98)00094-6

Cited by 4 publications

(2 citation statements)

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“…31 P NMR is a powerful instrument for measuring the vacuolar pH of intact plant cells [27]. When V. mungo seeds were allowed to germinate at 27 °C in darkness, the fresh weight of the axis increased until day 6, and that of cotyledons decreased concurrently with the growth of the axis [29].…”

Section: Resultsmentioning

confidence: 99%

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Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)

Okamoto

Yûki

Mitsuhashi

et al. 1999

European Journal of Biochemistry

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A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31 P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages. The activation of propapain [5,8] and human procathepsin B [13] has been intensively studied, and both were found to be activated by both intramolecular and intermolecular autocatalytic processings. Rat procathepsin B is completely processed to the mature form intermolecularly by mature cathepsin B and subsequent trimming by a dipeptidylpeptidase [14]. Procathepsin L [15,16] and procathepsin K [17] are also activated to the mature enzymes in a self-catalytic manner. In contrast, precursors of plant vacuolar cysteine proteinases, SH-EP [18] and aleurain [19], and an extracellular proteinase, EPB [20], are thought to be processed by other proteinases.SH-EP is a cysteine proteinase that plays a major role in the degradation of seed storage proteins accumulated in cotyledonary vacuoles of Vigna mungo seedlings [21]. It was previously reported that the enzyme is synthesized on membrane-bound ribosomes as a 45-kDa polypeptide, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a 2-kDa signal peptide. The intermediate is further processed to the 33-kDa mature enzyme via 39-and 36-kDa intermediates, and the conversion from the 36-kDa intermediate to the 33-kDa mature enzyme results in full activation [22]. Okamoto and Minamikawa [18] isolated a processing enzyme, designated VmPE-1, that is...

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Section: Resultsmentioning

confidence: 99%

“…Freshly harvested cotyledons of V. mungo seedlings were transferred into a NMR tube (10 mm in diameter), and the tube was filled with 30% D 2 O. 31 P NMR was conducted with a Lambda 400 NMR spectrometer operating at 161.7 MHz [27]. The spectra were measured at 2000 scans using a 45° pulse angle at 25.0 ± 0.1 °C.…”

Section: Methodsmentioning

confidence: 99%