Sticher L, Jones R L
Department of Plant Biology, University of California, Berkeley, California 94720.
Plant Physiol. 1991 Nov;97(3):936-42. doi: 10.1104/pp.97.3.936.
Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.
对导致大麦（Hordeum vulgare L. cv Himalaya）中出现多种形式α-淀粉酶（EC 3.2.1.1）的翻译后修饰进行了研究。当通过变性聚丙烯酰胺凝胶电泳分析时，大麦α-淀粉酶的分子量为43至44千道尔顿，但等电聚焦可将该酶分离为大量同工型。为了精确鉴定这些同工型，我们提出了一种基于其等电点（pI）的分类系统。pI约为5的α-淀粉酶，以前称为低pI或Amy1同工型，已被命名为HAMY1，而pI约为6的α-淀粉酶，称为高pI或Amy2，被命名为HAMY2。HAMY1和HAMY2的各个同工型通过其pI来鉴定。例如，大麦糊粉层合成并分泌的最酸性α-淀粉酶被命名为HAMY1(4.56)。大麦α-淀粉酶pI的一些差异源于该酶的翻译后修饰。我们报道了从大麦糊粉层和培养液中分离出一种因子，该因子可在体外修饰HAMY1同工型。该因子的分子量在30至50千道尔顿之间，它可以催化HAMY1(4.90)和HAMY1(4.64)分别转化为4.72和4.56同工型。该因子对HAMY1同工型的体外转化在pH值约为5时更有利，而在pH约为7时受到抑制。糊粉层和培养液中该因子的水平不受赤霉素处理组织的影响。来自大麦的淀粉酶修饰活性也会修饰从人唾液和猪胰腺中分离出的α-淀粉酶。还从Onozuka R10纤维素酶中分离出了一种可在体外修饰HAMY1同工型的活性。由于从大麦中分离出的活性会降低来自大麦、人唾液和猪胰腺的α-淀粉酶的pI，我们推测它是一种脱酰胺酶。
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