title: Importance of disulfide bridge formation on folding of phospholipase D from Streptomyces antibioticus.
abstract: The effects of redox conditions on the folding of phospholipase D (PLD) of Streptomyces antibioticus were investigated. Although the enzyme was very stable even in the presence of 1.0 M guanidinehydrochloride (Gdn-HCl), the coexistence of dithiothreitol (DTT) and Gdn-HCl inactivated the enzyme completely. The inactivated enzyme recovered its activity by dialysis in which DTT was removed prior to Gdn-HCl, whereas its activity was not recovered when Gdn-HCl was removed prior to DTT. In vitro protein synthesis was used for further analyses of the folding process. Active PLD was synthesized in the absence of DTT. The activity increased as the protein synthesis proceeded. In contrast, inactive PLD was synthesized in the presence of DTT. The inactive PLD could not be effectively activated by simple removal of the reductant, while incubation with Gdn-HCl and subsequent removal of DTT followed by that of Gdn-HCl was a much more effective method for the synthesis of active enzymes. From these results, it is suggested that: (i) PLD contains disulfide bridge(s), which is (are) necessary for maintaining its tertiary structure, (ii) correct formation of the disulfide bridge(s) is a critical step in the early stage of the (re)folding process, and (iii) the disulfide bridge(s) further facilitate the folding process, resulting in the synthesis of the active enzymes with the correct structure.
authors.affiliation: Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Iwasaki
authors.forename: T
authors.initials: T
authors.lastname: Nishiyama
authors.forename: Y
authors.initials: Y
authors.lastname: Kawarasaki
authors.forename: H
authors.initials: H
authors.lastname: Nakano
authors.forename: T
authors.initials: T
authors.lastname: Yamane