Kragh M, Mølbak L, Andersen S O
August Krogh Institute, Copenhagen University, Denmark.
Comp Biochem Physiol B Biochem Mol Biol. 1997 Sep;118(1):147-54. doi: 10.1016/s0305-0491(97)00055-2.
The urea-extractable proteins from calcified regions of intermoult cuticle of the lobster, Homarus americanus, have been separated by two-dimensional electrophoresis, showing that the extracts contain a large number of proteins. The major proteins have isoelectric points between 4 and 9, and their apparent molecular weights are between 5 and 30 kDa. Two of the proteins have been purified by a combination of ion-exchange chromatography, gel-filtration and RP-HPLC, and their complete amino acid sequences were determined by a combination of mass spectrometry and automated Edman degradation. Although they were purified from a single animal, both proteins were obtained as two isoforms. The isoforms of the smaller protein (HaCP4.6) differed only in a single position (phenylalanine/isoleucine), and the isoforms of the larger protein (HaCP11.6) differed in two positions (valine/isoleucine and glutamine/lysine). HaCP11.6 is N-terminally blocked by a pyroglutamate residue. Variants of an 18-residue motif are a characteristic feature of both sequences: it occurs twice in HaCP4.6 and four times in HaCP11.6. Comparison of the sequence to sequences published for cuticular proteins from other arthropods shows that the repeated motif is also present in proteins from the exoskeleton of the Bermuda land crab, Gecarcinus lateralis, but not in the single shrimp protein (Pandalus borealis) sequenced so far. The amino acid compositions of the lobster proteins are similar to that of flexible cuticles in locusts, but no convincing sequence similarities were found between the lobster proteins and cuticular proteins from locusts or other insects.
美洲螯龙虾（Homarus americanus）蜕皮间期表皮钙化区域的尿素可提取蛋白已通过二维电泳分离，结果表明提取物中含有大量蛋白质。主要蛋白质的等电点在4到9之间，其表观分子量在5到30 kDa之间。其中两种蛋白质通过离子交换色谱、凝胶过滤和反相高效液相色谱相结合的方法进行了纯化，并通过质谱和自动埃德曼降解相结合的方法确定了它们完整的氨基酸序列。尽管它们是从单只动物中纯化得到的，但两种蛋白质均以两种同工型形式获得。较小蛋白质（HaCP4.6）的同工型仅在一个位置（苯丙氨酸/异亮氨酸）存在差异，而较大蛋白质（HaCP11.6）的同工型在两个位置（缬氨酸/异亮氨酸和谷氨酰胺/赖氨酸）存在差异。HaCP11.6的N端被一个焦谷氨酸残基封闭。18个残基基序的变体是这两种序列的一个特征：它在HaCP4.6中出现两次，在HaCP11.6中出现四次。将该序列与已发表的其他节肢动物表皮蛋白序列进行比较，结果表明，百慕大陆地蟹（Gecarcinus lateralis）外骨骼中的蛋白质也存在这种重复基序，但在迄今为止测序的单一种类虾蛋白（北方长额虾，Pandalus borealis）中没有发现。龙虾蛋白的氨基酸组成与蝗虫柔性表皮的氨基酸组成相似，但在龙虾蛋白与蝗虫或其他昆虫的表皮蛋白之间未发现令人信服的序列相似性。
