Restoration of enzymatic activity in frog lens rho-crystallin

Research Project

Project/Area Number	06671756
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Ophthalmology
Research Institution	FUKUIMEDICAL SCHOOL
Principal Investigator	FUJII Yutaka FUKUI MEDICAL SCHOOL,DEPT.OF MEDICINE,ASSISTANT PROFESSOR, 医学部, 助教授 (80211522)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥2,200,000 (Direct Cost: ¥2,200,000) Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000) Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywords	rho-crystallin / molecular evolution / point mutation / restoration / カエル / アルド・ケト還元酵素 / 糖尿病性合併症 / ポリオール浸透圧説 / プロスタグランジンF合成酵素 / ポイントミューテーション
Research Abstract	From frog lens cDNA library, the clone (R7FF) encoding rho-crystallin was isolated. Expression vector pMR derived from R7FF was employed for the expression of mature-type rho-crystallin-II in E.coli.(HB101). N- and C-termini of the recombinant rho-crystallin were identical to those of rho-crystallin-II purified from frog lens. Among aldo-keto reductases such as prostaglandin F synthase, aldoes reductase, and aldehyde reductase, 55-threonine is specific for rho-cystallin and tyrosine is conserved among all of other aldo-keto reductases. Therefore, 55-tyr-rho-crystallin mutant was prepared, the mutant showed a aldo-keto reductase activity. It was demonstrated that the restoration of enzymatic activity in rho-crystallin was achieved by the single point mutation of 55-threonine to tyrosine. When 9,10-phenanthrenequinone was used as substrate at pH 6.5, the specific activity of the mutant was determined to be 0.63 units/mg protein at 30ﾟC.Reduction of glucose was also observed in the mutant enzyme. The activity of the mutant enzyme corresponded to that of aldose reductase which contribute to formation of diabetic complications. During the molecular evolution of rho-crystallin, point mutation of 55-tyrosine to threonine may occurred to avoid aldose reductase like activity.