Complete amino acid sequence of the α-amylase/subtilisin inhibitor from barley

Svendsen, Ib; Hejgaard, Jørn; Mundy, John

doi:10.1007/bf02907994

Cited by 61 publications

(57 citation statements)

References 32 publications

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“…Triumf), each from Carlsberg Maltings. The protein concentrations were determined by amino acid analysis of acid hydrolysates (6 N HCI, 110°C, 24 h) using an LKB model Alpha Plus amino acid analyzer, or spectrophotometrically at 280 nm using e = 2.6 x 104 M -~ .cm -t for BASI [1,7] and e = 1.13 x 105 M -l .cm -1 for AMY2 [161. Insoluble blue starch was from Pharmacia, Uppsala, Sweden.…”

Section: Methodsmentioning

confidence: 99%

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Stopped‐flow kinetic studies of the reaction of barley α‐amylase/subtilisin inhibitor and the high pI barley α‐amylase

et al. 1995

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Key words." Reaction mechanism; Bifunctional a-amylase/ serine protease inhibitor; Stopped-flow fluorescence kinetics; Substrate-mediated inhibitor displacement; Barley; High pI a-amylase dimeric 0.19 a-amylase inhibitor from wheat [14]. No a-amylase inhibitor reaction has been thoroughly, kinetically investigated, but those of the red kidney bean a-amylase inhibitor and porcine pancreatic a-amylase have been partially characterized by Wilcox and Whitaker [15], who reported the formation of a 1 : 1 complex in a two-step reaction path.In the present study the kinetics of the formation of complexes between BASI and AMY2 have been investigated in order to expand our knowledge of the inhibition mechanism of proteinaceous a-amylase inhibitors. The results were in accordance with a two-step reaction model, the equilibrium and rate constants of which were determined from a combination of stopped-flow and substrate displacement kinetic data. The BASI-AMY2 and the red kidney bean a-amylase inhibitor--~-amylase reactions apparently differ markedly in their second reaction step, so that, in spite of similar overall dissociation constants, they seem to belong to different classes. In contrast to the red kidney bean inhibitor, which is slow reacting, BASI was found to be a fast reacting, tight-binding inhibitor. Materials and methods

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Section: Methodsmentioning

confidence: 99%

“…Barley seeds contain a bifunctional a-amylase/subtilisin inhibitor, (BASI) of Mr 19,865 [1,2]. Based on sequence similarities it belongs to the soybean trypsin inhibitor (Kunitz) family [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Stopped‐flow kinetic studies of the reaction of barley α‐amylase/subtilisin inhibitor and the high pI barley α‐amylase

et al. 1995

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“…In contrast to these inhibitors, PKI-2 appears to have a somewhat broad specificity, with limited but significant activity against both trypsin and chymotrypsin. Three Kunitz-type subtilisin inhibitors from monocot seeds, barley (22), wheat (9), and rice (22), have been characterized and sequenced. The inhibitors from barley and wheat are bifunctional and possess activity against insect amylases (3).…”

Section: Assays Of Proteinase Inhibitionmentioning

confidence: 99%

Two Kunitz-Type Proteinase Inhibitors from Potato Tubers

Walsh

Twitchell²

1991

Plant Physiol.

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Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et a!. [1989] (19,20). In this paper, we describe two serine proteinase inhibitors from potato tubers that have differing proteinase inhibitory profiles but are both Kunitztype inhibitors based on amino-terminal sequence homologies. One closely resembles the previously described cathepsin D inhibitor (10). The other is an inhibitor of the microbial proteinase subtilisin and appears to be the same as the abundant 22 kD protein isolated by Suh et al. ( 19). This is the first Kunitz-type inhibitor of a microbial proteinase to be identified in a source other than from small grains. MATERIALS AND METHODSPotato (Solanum tuberosuim) tubers (var Russet Burbank) were purchased from a local market. Bovine trypsin, bovine Mono S 5/5 column equilibrated in 25 mm sodium acetate (pH 5.0) and using a 0 to 0.5 M NaCl gradient over 30 min with a flow rate of 1 mL/min. Reverse phase HPLC was performed with a Vydac C4 4.5 x 300 mm column equilibrated in 0. 1% TFA and eluted with a gradient of 0 to 35% acetonitrile over 8 min, then 35 to 65% acetonitrile over 30 min at a flow rate of 1 mL/min. Peaks were collected and lyophilized prior to SDS-PAGE analysis and amino terminal sequencing.15

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“…The elution by a linear sodium chloride gradient exhibited 5 distinct major peaks of proteins (data not shown). Analysis of the protein components of these peaks by SDS-PAGE revealed that in one peak (called III), at least 80% of the total amount of the protein C was co-eluted with the barley a-amylase/subtilisin inhibitor, BASI (20). This "III" fraction, consisting mainly of the two proteins, was further purified by CM-52 cation exchange chromatography as shown in Figure 1.…”

Section: Isolation Of Barley Protein Cmentioning

confidence: 99%