ISOLATION, PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN Α-Amylase FROM MALTED BARLEY

MacGregor, A. W.

doi:10.1002/j.2050-0416.1977.tb06424.x

Cited by 26 publications

(11 citation statements)

References 17 publications

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“…Scanning electron micrographs (Figs. 3 and 4) and previous studies ( 17,19) demonstrated that the equatorial groove of a starch granule was more susceptible to attack by a-amylases than were the broad, flat surfaces. Hydrolysis by a-amylases alone appears to be dependent upon penetration of the enzymes at the equatorial groove.…”

Section: Discussionmentioning

confidence: 78%

Degradation of Native Starch Granules by Barley α-Glucosidases

Sun¹,

Henson²

1990

Plant Physiol.

132

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The initial hydrolysis of native (unboiled) starch granules in germinating cereal kemels is considered to be due to a-amylases. We report that barley (Hordeum vulgare L.) seed a-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant a-amylase isozymes. Two a-glucosidase charge isoforms were used individually and in combination with purified barley a-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl a-glucosidase was combined with either high or low pl a-amylase. Synergism was also found when low pi a-glucosidase was combined with a-amylases. Scanning electron micrographs revealed that starch granule degradation by a-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of a-glucosidases and a-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by a-amylase alone. As the presence of a-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the a-glucosidases to hydrolyze glucosidic bonds other than a-1,4-and a-1,6-that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by a-amylases. Since both a-glucosidase and a-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.Although the first discovered enzyme is usually considered to be diastase (23) Evidence of a non-a-amylase starch hydrolyzing factor in malted wheat was reported by Blish et al. (4). They found a 'raw starch catalyst,' a factor separate and distinct from aamylase, which greatly increased a-amylase hydrolysis of native starch granules but did not significantly alter the activity on soluble starch. Work with the wheat system was not pursued; hence, the activating factor has not been elucidated. However, a marked supplementing effect of Aspergillus oryzae amylase preparations on the hydrolysis of corn starch by pancreatic a-amylase has been reported (2). The supplementing factor in the mold was later traced to the presence of aglucosidase (maltase) (21). Similar effects of a-glucosidase have also been termed 'amylase activation' and 'complementary action.' As no enzymes other than a-amylases have been demonstrated to be capable ofattacking native starch granules from tissues of higher plants, these a-glucosidase effects have been attributed to its degradation of maltose, which is both an end product and an inhibitor of a-amylolysis.We report here the identification of a-glucosidase (EC 3.2.1.20) as the enzyme responsible for the a-amylase 'activation' in germinating barley and that this supplementing effect is not just due to simple removal of maltose. We demonstr...

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Section: Discussionmentioning

confidence: 78%

Degradation of Native Starch Granules by Barley α-Glucosidases

Sun¹,

Henson²

1990

Plant Physiol.

132

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“…The protein sequence data for a-amylase 2, a high pI isozyme, showed that it corresponds to the partial-length a-amylase cDNA clone pHV 19 described by CHANDLER et al (4) and thus most probably also to the clone 103 characterized by MUTHUKRISHNAN et al (17,18). In the literature the low and high pI isozymes have been designated respectively by I and 2 (12)(13)(14). Other workers named them the group A and B isozymes (4, 7) specified by the a-Amy 2 and a-Amy 1 loci (1).…”

Section: Discussionmentioning

confidence: 99%

“…The barley a-amylases were isolated as de- (7,12,14). The amino acid compositions of the a-amylase 1 and 2 preparations (Table I) clearly differed from each other only in their serine, alanine and methionine contents.…”

Section: Isolation and Characterization Of Aamylase 1 Andmentioning

confidence: 99%

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Partial amino acid sequences of α-amylase isozymes from barley malt

Svensson

Mundy

Gibson

et al. 1985

Carlsberg Res. Commun.

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“…This agrees with HEBERT (9) which also described the existence of relatively high concentrations of acid glycoproteins in beer and demonstrated that these components are of special significance for the foam properties of beer. The observed heterogeneity of this acid protein fraction might, however, also originate from its intrinsic heterogeneity in the barley plant since it has frequently been observed that such well defined enzymes as peroxidases (24) esterases (6) and namylases (19) in barley and malt are present as isoenzymes covering wide ranges of isoelectric points. Further investigations of this possibility will have to await the isolation of the Z-protein.…”

Section: Discussionmentioning

confidence: 99%

Fractionation and characterization of beer proteins

Sørensen

Ottesen

1978

Carlsberg Res. Commun.

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A combination of large scale gel filtration with preparative isoelectric focusing and ion exchange chromatography has been used for fractionation of beer proteins. From gel filtration on Sephadex G-150, a high molecular weight fraction, eluting close to the void volume of the column, a fraction appearing corresponding to a molecular weight of 44,000 and a fraction containing rather low-molecular weight (about 10,000) components were obtained and used for preparative isoelectric focusing in the pH-range 3.5-10. The high-molecular weight fraction was rich in carbohydrate and cross-reacted with yeast antibodies. After preparative isoelectric focusing the amino acid composition of the isolated subfractions resembled that of yeast cell wall components. Preparative isoelectric focusing of the two fractions with molecular weight about 44,000 and 10,000 revealed the presence of two classes of components based on their amino acid composition. One class, which seemed to be present in low concentration in nearly all isolated subfractions, had an amino acid composition resembling that of barley glutelins. In the fraction with molecular weight about 44,000 another class of components -having an amino acid composition resembling barley albumins and globulins -Were observed in the region with isoelectric points of about pH 4-5. They cross-reacted immunologically with antibodies against soluble barley proteins. This class of components could also be separated from the glutelin-like constituents by ion exchange chromatography. However, they could not be completely separated from carbohydrate by the fractionation procedures employed. Carbamylation experiments demonstrated that approx, half of the e-amino groups of lysine residues of these proteins were blocked. Furthermore, partial amino acid sequence determination by the Edman procedure revealed a strong heterogeneity with respect to N-terminal amino acid residues. These observations are consistent with a suggestion that a large part of the beer proteins might be polypeptide chains crosslinked by carbohydrates.

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ISOLATION, PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN Α-Amylase FROM MALTED BARLEY

Cited by 26 publications

References 17 publications

Degradation of Native Starch Granules by Barley α-Glucosidases

Degradation of Native Starch Granules by Barley α-Glucosidases

Partial amino acid sequences of α-amylase isozymes from barley malt

Fractionation and characterization of beer proteins

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