2000
DOI: 10.1046/j.1432-1327.2000.01201.x
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Characterization of potato proteinase inhibitor II reactive site mutants

Abstract: Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (K i 0.4 nm) and chymotrypsin (K i … Show more

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Cited by 43 publications
(33 citation statements)
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References 43 publications
(82 reference statements)
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“…They are known to be nonspecific protease inhibitors that can effectively inhibit both trypsin and chymotrypsin [19,23,24]. The lower sensitivity for detecting these inhibitors in trypsin-treated Z-Phe-Arg-MCA gel in comparison with Boc-Phe-Ser-Arg-MCA gel is in good agreement with kinetic data reported previously [13,14,25].…”
Section: Resultssupporting
confidence: 86%
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“…They are known to be nonspecific protease inhibitors that can effectively inhibit both trypsin and chymotrypsin [19,23,24]. The lower sensitivity for detecting these inhibitors in trypsin-treated Z-Phe-Arg-MCA gel in comparison with Boc-Phe-Ser-Arg-MCA gel is in good agreement with kinetic data reported previously [13,14,25].…”
Section: Resultssupporting
confidence: 86%
“…2). Under reducing conditions, the original PCI is known to have a molecular mass of 11 kDa as a monomer that is capable of binding to chymotrypsin or to trypsin [18,19]. Since the present technique preserved the inhibitory activity without heat treatment or reducing reagents, it is possible that the higher molecular mass forms observed in this case were due to aggregation of smaller inhibitor forms.…”
Section: Resultsmentioning
confidence: 92%
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“…A characteristic feature of PI-II proteins is that they contain one to several sequence repeats, which code for one precursor. Proteolytic cleavage of these precursors at specific linker sites results in several single or multidomain PIs with different properties (Atkinson et al, 1993;Beekwilder et al, 2000;Horn et al, 2005;Tamhane et al, 2007), which could explain the absence of more than two bands when silencing genes encoding the two-domain proteins SPI2a and SPI2b. It is possible that other peptides with low activity or concentration were not detected or resolved with the method that we used and that there are in fact more than three SPI peptides missing in the (A) Mean 6 SE relative transcript abundance of SPI1, SPI2a, and SPI2b in leaves of wild-type plants and of transgenic lines expressing an irconstruct specific for SPI2a and SPI2b (irSPI2a+b) or a construct specific for SPI1, SPI2a, and SPI2b (irSPI1/2a+b) (n = 7).…”
Section: Spi2a and Spi2b Are Strong Inhibitors Of Subtilisin But Accomentioning
confidence: 99%
“…Secondary contacts not involving the reactive-site loop in Domain II of two-domain Pot II family inhibitors may be of critical importance to determining proteinase inhibition specificity, as suggested by the results of site-directed mutagenesis studies on PI-II from potato (66). In PI-II, the capacity for trypsin inhibition in Domain II could not be transferred to Domain I by mutation of the P1 residue, the P2-P1-P1Ј sequence, or even the entire stretch of sequence from P2 to P10Ј.…”
Section: Table IImentioning
confidence: 99%