Study on isozymes of isocitrate dehydrogenase from a psychrophilic bacterium, Vibrio sp. strain ABE-1 好冷菌Vibrio ABE-1株のイソクエン酸脱水素酵素アイソザイムに関する研究

博士論文

It was preciously found in our laboratory that an obligately psychrophilic Vibrio sp. strain　ABE-1 had two isozymes of NADP＋ -specific isocitrate dehydrogenase ［IDH； EC　1．1．1．42］ which were rnarkedly different in their temperature dependency and so on [Ochiai et al.（1979）， J. Biochem. , 86；377－384］． In order to elucidate their physiological　significance and their evolutionary relationship, it was absolutely necessary to purify these　isozymes. However, these isozymes could not be purified so far. Then, firstly I attempted to develop the purification procedures of the enzymes, and subsequently biochemical and physical　properties of the enzymes were investigated. Next catalytic properties of the isozymes were revaluated. Finally, their structural relation was evaluated by comparison of the immunoreactivities, the amino acid sequences at the amino terminals, and the complete nucleotide sequences （Part　ll）.　 Application of the hydrophobic chromatography in an early step of the procedure resulted in complete separation of the two isozymes from each other followed by being successful in purification to electrophoretically homogeneous state. Apparent molecular weights of the isozymes　estimated by gel filtration were 88,100 for isozyme－I （IDH－I） and 80,500 for isozyme－II 　（IDH - II）, whereas 49,100 for IDH－I and 79,500 for IDH－II by gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that IDH－I was dimer and IDH－II was a single polypeptide. The isoelectric points of IDH－I and －II were found to be 4．9 and 5．2, respectively . The absorption maximum and shoulder in ultraviolet region of the isozymes were observed at 278 nm and 292 nm, and values of El(I)cm(％) at 280 nm were 6.15 for IDH－1 and 4.01 for IDH－II． Although the two isozymes were similar in amino acid compositions with slight differences in the contents of nonpolar and hydroxyl amino acids, the NH2－terminal amino acid sequences were quite different． Moreover， Ouchterlony double immunodiffusion and immunotitration of the each isozyme with the respective counter antibody revealed that the isozymes were different in immunochemical property from each other. There observed remarkable differences not only in thermostability but also in thermal inactivation mode between the isozymes, that is, IDH－I was inactivated linearly in the incubation above 45℃,　whereas IDH－II was inactivated biphasicaly above 20℃. Some nucleotides and metabolites in glycolysis, tricarboxylic acid cycle and glyoxylate bypass acted as inhibitors to the IDH isozymes. Potassium ion was found to be the best monovalent cation for activities of the both isozymes, even though the range of optimum concentration of K＋ for IDH-II was broader than that for IDH-I. Additionally, the isozymes were different in kinetic and thermodynamic parameters. From these results, I conclude that IDH－II is more adaptive than IDH-I under the condition which is low temperature and high salt concentration.