Robertson J G, Desai P R, Kumar A, Farrington G K, Fitzpatrick P F, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 18802.
J Biol Chem. 1990 Jan 15;265(2):1029-35.
Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.
通过反相高压液相色谱法分离并测序了来自牛多巴胺β-羟化酶的58个胰蛋白酶和金黄色葡萄球菌V8蛋白酶产生的肽段。将这些肽段序列与从克隆的cDNA获得的牛和人多巴胺β-羟化酶的推导氨基酸序列进行了比较。牛肽段序列与来自牛cDNA的序列有5个差异，其中4个变化可由DNA中的单个碱基变化来解释。牛酶的N端序列分析表明它含有两个N端，其中一个比另一个长3个氨基酸，起始序列为Ser-Ala-Pro。从牛和人cDNA推导的氨基酸序列分别长19和25个氨基酸，这些额外的氨基酸代表前导肽序列。两个牛肽段序列含有糖基化位点并对碳水化合物残基呈阳性检测，另外两个含有糖基化位点的共有序列但在碳水化合物检测中呈阴性。与牛cDNA中的7个和人cDNA中的8个相比，牛酶含有6个色氨酸。蛋白质和牛cDNA各含有24个酪氨酸，而人cDNA中有26个。这些数字表明真正的ε1% 280 = 8.95，因此，它比先前确定的值低28%。数据还确定了5个含组氨酸的区域，这些区域可能参与活性位点的Cu2+配位。
