Lebherz H G, Leadbetter M M, Bradshaw R A
J Biol Chem. 1984 Jan 25;259(2):1011-7.
Two different isoenzymes of fructose-P2 aldolase can be resolved by chromatography of crude spinach leaf extracts on DEAE-cellulose columns. The acidic isoenzyme comprises about 85% of the total leaf aldolase activity. The two forms differ in primary structure as judged by their distinctive amino acid compositions, tryptic peptide patterns, and immunological properties. Only the acidic isoenzyme was detected in extracts of isolated chloroplasts, suggesting that this molecule represents the chloroplast form of spinach leaf aldolase while the basic isoenzyme is of cytosolic origin. The cytosolic (basic) isoenzyme and chicken aldolase A4 are similar in the following respects. 1) They have similar specific catalytic activity (10-15 units/mg); 2) they are both highly sensitive to inactivation by very limited digestion with bovine pancreatic carboxypeptidase A; 3) they both have subunit molecular weights of 40,000; 4) they both have derivatized (blocked) NH2-terminal structures; 5) they are both resistant to thermal denaturation at 50 degrees C; and 6) they both regain catalytic activity following reversible denaturation at pH 2.3 or in 5.8 M urea. Also, the cytosolic aldolase cross-reacted immunologically with the single aldolases present in spinach seeds and in wheat germ. Further, this isoenzyme readily "hybridized" with chicken aldolase A4 in vitro. These observations demonstrate the close homology between the cytosolic aldolases derived from plant and animal origins. The chloroplast aldolase had a specific catalytic activity of about 8 units/mg and, like its cytosolic counterpart, was severely inactivated by limited digestion with carboxypeptidase A. However, this isoenzyme was distinct from the cytosolic aldolase in the following characteristics: 1) its "small" subunit size (Mr congruent to 38,000); 2) its underivatized NH2-terminal structure; 3) its high sensitivity to thermal denaturation at 50 degrees C; and 4) its inability to refold into an enzymatically active conformation following denaturation at pH 2.3 or in 5.8 M urea. The distinctive properties of the chloroplast aldolase may be expected for an enzyme which is synthesized as a higher molecular weight precursor on cytosolic polysomes and is then proteolytically processed to the "mature" form during its migration into the chloroplast organelle.
通过在DEAE - 纤维素柱上对菠菜叶粗提物进行层析，可以分离出两种不同的果糖 - P2醛缩酶同工酶。酸性同工酶约占叶片总醛缩酶活性的85%。根据它们独特的氨基酸组成、胰蛋白酶肽图谱和免疫特性判断，这两种形式在一级结构上有所不同。在分离的叶绿体提取物中仅检测到酸性同工酶，这表明该分子代表菠菜叶醛缩酶的叶绿体形式，而碱性同工酶则起源于细胞质。细胞质（碱性）同工酶与鸡醛缩酶A4在以下方面相似：1）它们具有相似的比催化活性（10 - 15单位/毫克）；2）它们都对用牛胰羧肽酶A进行的非常有限的消化导致的失活高度敏感；3）它们的亚基分子量均为40,000；4）它们都具有衍生化（封闭）的NH2 - 末端结构；5）它们在50℃下都对热变性具有抗性；6）它们在pH 2.3或5.8 M尿素中可逆变性后都能恢复催化活性。此外，细胞质醛缩酶与菠菜种子和小麦胚芽中存在的单一醛缩酶发生免疫交叉反应。此外，这种同工酶在体外很容易与鸡醛缩酶A4“杂交”。这些观察结果证明了源自植物和动物的细胞质醛缩酶之间的密切同源性。叶绿体醛缩酶的比催化活性约为8单位/毫克，并且与其细胞质对应物一样，在用羧肽酶A进行有限消化时会严重失活。然而，这种同工酶在以下特征上与细胞质醛缩酶不同：1）其“小”亚基大小（Mr约为38,000）；2）其未衍生化的NH2 - 末端结构；3）其在50℃下对热变性的高敏感性；4）其在pH 2.3或5.8 M尿素中变性后无法重新折叠成具有酶活性的构象。叶绿体醛缩酶的独特性质可能是预期的，因为该酶作为较高分子量的前体在细胞质多核糖体上合成，然后在其迁移到叶绿体细胞器的过程中被蛋白水解加工成“成熟”形式。
