A cold acclimation protein with refolding activity on frozen denatured enzymes.
We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100 degrees C, cryopreservation at -20 degrees C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12 degrees C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82x10(-10), 4.35x10(-9), 8.98x10(-12), and 3.05x10(-11), respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.
Kawahara
H
H
Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka, Japan. kawahara@ipcku.kansai-u.ac.jp
Koda
N
N
Oshio
M
M
Obata
H
H
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D018834:Chaperonin 60; D003080:Cold Temperature; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D020636:Pantoea / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D018834:Chaperonin 60; D004798:Enzymes; D006360:Heat-Shock Proteins; D007770:L-Lactate Dehydrogenase