EXTRACTION AND ISOLATION OF ANTIFREEZE PROTEINS FROM WINTER RYE (SECALE-CEREALE L) LEAVES
EXTRACTION AND ISOLATION OF ANTIFREEZE PROTEINS FROM WINTER RYE (SECALE-CEREALE L) LEAVES
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DOI：
10.1104/pp.104.3.971
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发表时间：
1994-03-01
期刊：
影响因子：
7.4
通讯作者：
YANG, DSC
中科院分区：
文献类型：
--
作者：
HON, WC;GRIFFITH, M;YANG, DSC
冷驯化冬黑麦（Secale cereale L.）cv Musketeer）叶先前显示出抗冻活性。本研究的目的是鉴定和表征存在于质外体提取物中的个体抗冻蛋白。最高的蛋白质浓度和抗冻活性时，叶质外体提取抗坏血酸和CaCl 2或MgSO 4。七个主要的多肽纯化，从这些提取物的一维十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳在非还原条件下。5个较大的多肽，19，26，32，34和36 kD，表现出显着水平的抗冻活性，而11和13 kD的多肽只显示出较弱的活性。这些多肽迁移较高的表观分子量SDS凝胶处理后，与0.1 M二硫苏糖醇，这表明存在分子内二硫键。明显减少的二硫键没有消除抗冻活性的多肽，含有分子内二硫键，并表现出显着水平的抗冻活性。这些多肽的氨基酸组成是相似的，因为它们都相对富含残基Asp/Asn、Glu/Gln、Ser、Thr、Cry和Ala;除了26-kD多肽外，它们都缺乏His，并且它们含有高达5%的Cys残基。这些多肽与其他含半胱氨酸的抗冻蛋白从鱼类和昆虫的抗血清进行了检查，并没有共同的表位被检测到。我们的结论是冷驯化冬黑麦叶产生多种多肽与抗冻活性，似乎是不同的鱼和昆虫产生的防冻剂。
Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Cry, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystein-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.