Identification and Partial Characterization of High Mr Neutral Proteinases from 4-day Germinated Barley Seed

Wrobel, Radoslawa; Jones, B. L.

doi:10.1006/jcrs.1993.1049

Cited by 17 publications

(20 citation statements)

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“…3) or watertreated (data not shown) protoplasts have much less Cp activity than those isolated from GA,-treated protoplasts. Increases in CP activity following GA, treatment have previously been reported for secreted barley proteases Ho, 1988, 1990), proteases of unknown subcellular location (Wrobel and Jones, 1993), and aleurain (Holwerda et al, 1990). Our data represent the first clear demonstration that CP activity in barley aleurone PSV is increased by GA, treatment.…”

Section: Discussionsupporting

confidence: 43%

“…Most of these enzymes, however, are secreted proteases. Among these are at least 4 CPs Ho, 1988, 1990;Poulle and Jones, 1988;Wrobel and Jones, 1992), 6 carboxypeptidases (Rocher et al, 1994), and 10 neutra1 proteinases (Wrobel and Jones, 1993). Only a few proteases are known to be retained within the aleurone.…”

supporting

confidence: 45%

“…Although severa1 AP activities have been found in barley (Wrobel and Jones, 1993), the only well-characterized AP is HvAP. This protease has previously been shown to be in the embryo, aleurone layer, testa, and pericarp of the barley grain, with little in the starchy endosperm (Tormakangas et al, 1994).…”

Section: Discussionmentioning

confidence: 43%

“…The assay was based on the zymogram procedure of Wrobel and Jones (1993), whereby proteolytic activity is measured following nondenaturing PAGE. Briefly, 10% ( w / v ) acrylamide gels were prepared with acid-denatured hemoglobin added to the resolving gel (250 mM Tris-HC1, pH 9.1) to a final concentration of 0.032% (w/v).…”

Section: Assays For Proteolytic Activitymentioning

confidence: 43%

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Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

Bethke¹,

Hillmer²,

Jones³

1996

Plant Physiol.

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Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). W e developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare 1. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. lsolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. W e used isolated PSV to identify and characterize PSV-associated proteolytic activities. lsolated PSV contained cysteine proteases and aspartic proteases (APs). Cibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vurgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. lmmunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. W e conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.

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Section: Discussionsupporting

confidence: 43%

supporting

confidence: 45%

Section: Discussionmentioning

confidence: 43%

Section: Assays For Proteolytic Activitymentioning

confidence: 43%

See 2 more Smart Citations

Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

Bethke¹,

Hillmer²,

Jones³

1996

Plant Physiol.

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“…It has been widely reported that cysteine and metallo proteinases, the same proteinases indicated for barley adjunct wort from our research, had a high correlation to wort FAN levels in all-malt wort 7,8,10,12,13,23 . These similar consequences from two different production processes seem to conflict at first sight.…”

Section: Why Are Proteinase Activities That Contribute To Fan Productmentioning

confidence: 44%

The Effect of Barley Adjuncts on Free Amino Nitrogen Contents in Wort

Yano

Tsuda

Imai

et al. 2008

Journal of the Institute of Brewing

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: Free amino nitrogen (FAN) and other low‐molecular‐weight nitrogen compounds (LNC) are highly important as nutrients for yeast. Many different types of low‐malt beer exist around the world, some of which are produced with barley as an adjunct. In these cases, inhibitors contained in barley are known to influence the amount of LNC in wort. Accordingly, it is important to investigate which proteinase class is key in producing these compounds. By investigating the relationship between the FAN contained in wort produced from malt and barley (barley adjunct wort) and malt proteinase activity, it was found that cysteine proteinase and 1,10‐orthophenanthroline (O‐Phen)‐inhibitable metallo proteinases had a significant correlation to the barley adjunct wort FAN levels. In addition, the relationship between malting conditions and these proteinase activities was investigated and the conditions defined for maximal production of proteinases as follows: steeping degree, 50%; germination temperature, 12°C; germination days, 6 days; water spray, 3 times and concentration of gibberellic acid, 10 mg/kg (barley).

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Limit Dextrinase - Does Its Malt Activity Relate to Its Activity During Brewing?

McCafferty

Jenkinson

Brosnan

et al. 2004

Journal of the Institute of Brewing

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Limit dextrinase (EC 3.2.1.142) hydrolyses α‐1,6 glucosidic bonds in amylopectin and branched dextrins. Measurement of limit dextrinase activity during fermentation of unboiled wort at the pH of the wort has shown that its activity increases almost 10 fold during the first 10–15 h of fermentation and this increase in activity is unaffected by the presence of leupeptin, a cysteine protease inhibitor. The increase in activity seen when assays were carried out at pH 5.5 was much smaller and was reduced by leupeptin. The activity of limit dextrinase declined slowly during the latter part of the fermentation. It was established that the optimum pH for rapid extraction and assay of malt limit dextrinase in the absence of a reducing agent is approximately 4.5, but in the presence of dithiothreitol, at pH 5.5, activities 2–3 times higher can be obtained after 5 h extraction (600–700 mU/g dry weight). Limit dextrinase activities after 1 h extraction at mashing temperatures were below 20 mU/g dry weight if the mash pH was below 5.0. It is concluded that at pHs below 5.0, where limit dextrinase activity is below its optimum for activity, limit dextrinase activity increases due to dissociation of the inhibitor/enzyme complex. The protection from mashing temperatures of 65°C afforded by the inhibitor is lost at these lower pHs.

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Identification and Partial Characterization of High Mr Neutral Proteinases from 4-day Germinated Barley Seed

Cited by 17 publications

References 0 publications

Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

The Effect of Barley Adjuncts on Free Amino Nitrogen Contents in Wort

Limit Dextrinase - Does Its Malt Activity Relate to Its Activity During Brewing?

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