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Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp
Authors:S Nishikawa  H Morioka  K Fuchimura  T Tanaka  S Uesugi  E Ohtsuka  M Ikehara
Affiliation:1. Trauma Research Department, Swedish Medical Center, 501 E Hampden, Englewood, CO 80113, USA;2. Trauma Research Department, St. Anthony Hospital, 11600 W 2nd Pl, Lakewood, CO 80228, USA;3. Trauma Research Department, Medical City Plano, 3901 W 15th St, Plano, TX 75075, USA;4. Trauma Research Department, Penrose Hospital, 2222 N Nevada Ave, Colorado Springs, CO 80907, USA;5. Trauma Research Department, Research Medical Center, 2316 E Meyer Blvd, Kansas City, MO 64132, USA;6. Trauma Research Department, Wesley Medical Center, 550 N Hillside St, Wichita, KS 67214, USA;7. Department of Molecular Biology, Rocky Vista University, 8401 S Chambers Rd, Parker, CO 80134, USA;8. Department of Anesthesiology, St. Anthony Hospital, Lakewood, CO 80228, USA;9. Department of Anesthesiology, Wesley Medical Center, Wichita, KS 67214, USA;10. Department of Anesthesiology, Medical City Plano, Plano, TX 75075, USA;11. Department of Critical Care, Research Medical Center, Kansas City, MO 64132, USA;1. Department of Biochemistry and Molecular Biology, University of California, Riverside, USA;2. Neuroscience Graduate Program, University of California, Riverside, USA;3. Center for Glial-Neuronal Interactions, University of California, Riverside, USA;4. Division of Biomedical Sciences, School of Medicine, University of California, Riverside;1. Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China;2. Department of Nephrology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China
Abstract:Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.
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