␣-Synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only ␣-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of ␣-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human ␣-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of ␣-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of ␣-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of ␣-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.

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Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(1) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.