Schlatter D, Kriech O, Suter F, Zuber H
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, Zürich, Schweiz.
Biol Chem Hoppe Seyler. 1987 Nov;368(11):1435-46. doi: 10.1515/bchm3.1987.368.2.1435.
L-lactate dehydrogenase of the psychrophilic bacterium B. psychrosaccharolyticus was isolated by a three-step procedure and its total amino-acid sequence determined by automated Edman degradation. The protein consists of 318 amino-acid residues and its calculated molecular mass is 35,254 Da. Most of the primary structure could be established by sequencing large peptide fragments obtained by chemical cleavages, namely with BNPS-skatole and with CNBr. Further fragmentations of two tryptophan peptides with the endoproteinase Lys-C and with diluted HCl resulted in shorter overlapping peptides, the analysis of which completed the sequence. The C-terminal sequence Glu-Gln was established by carboxypeptidase A experiments and was then verified by the analysis of short C-terminal tryptic and chymotryptic peptides. The first lactate dehydrogenase sequenced so far of a psychrophilic bacillus shows sequence homologies between 60% and 75% to the enzymes from the mesophilic B. megaterium and B. subtilis and the thermophilic B. stearothermophilus, B. caldolyticus and B. caldotenax. Within the 50 N-terminal residues, three additional sequences could be included in our comparisons. In this part of the molecule, sequence homologies between 56% and 74% were calculated.
嗜冷性细菌嗜糖芽孢杆菌的L-乳酸脱氢酶通过三步法进行分离，并通过自动埃德曼降解法确定其完整氨基酸序列。该蛋白质由318个氨基酸残基组成，计算得到的分子量为35,254道尔顿。大部分一级结构可通过对化学裂解（即使用BNPS-粪臭素和溴化氰）得到的大肽段进行测序来确定。用内肽酶Lys-C和稀盐酸对两个色氨酸肽进行进一步裂解，得到了更短的重叠肽段，对这些肽段的分析完成了序列测定。通过羧肽酶A实验确定了C末端序列Glu-Gln，随后通过对短的C末端胰蛋白酶肽段和糜蛋白酶肽段的分析进行了验证。到目前为止测序的首例嗜冷芽孢杆菌乳酸脱氢酶与嗜温性巨大芽孢杆菌、枯草芽孢杆菌以及嗜热性嗜热脂肪芽孢杆菌、嗜热解糖芽孢杆菌和嗜热栖热芽孢杆菌的酶之间的序列同源性在60%至75%之间。在50个N末端残基范围内，我们的比较中还纳入了另外三个序列。在分子的这一部分，计算得到的序列同源性在56%至74%之间。
