Dissociation of the cellulosome of Clostridium thermocellum in the presence of ethylenediaminetetraacetic acid occurs with the formation of truncated polypeptides
- Univ. of Georgia, Athens, GA (United States)
The cellulosome of Clostridium thermocellum JW20 consists of 14-26 different polypeptides as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intact cellulosome hydrolyzes crystalline cellulose in the presence of Ca{sup 2+} and thiols. This activity is inhibited by ethylenediaminetetraacetic acid (EDTA). Ca is incorporated into the cellulosome and is tightly bound as demonstrated using {sup 45}Ca added to the growth medium. Upon incubation in 50 mM Tris (pH 7.5), 0.1 M NaCl, and 5 mM EDTA at 37{degrees}C, C is released form the cellulosome, which disintegrates into polypeptides. The SDS-PAGE pattern of cellulosomal polypeptides is remarkably different after the EDTA treatment when compared to this pattern of untreated cellulosomes. Polypeptide bands corresponding to molecular masses of 160, 98, 76, and 54 kDa disappear, and new bands of masses 150, 132, 91, 71, 57,and 46 kDa appear. N-terminal analyses of the 98, 76, 91, and 71 kDa polypeptides show that the 91 and 71 kDa polypeptides are truncated products of the 98 and 76 kDa polypeptides, respectively. The 76 and 71 kDa polypeptides correspond to CelS. The 71 kDa polypeptide is formed from the 76 kDa polypeptide during the EDTA treatment, by a cleavage that occurs at asparagine residue 681. It involves the removal of 60 amino acid residues from the C-terminal end. All catalytic subunits so far characterized contain an asparagine residue corresponding to residue 681 of CelS. This residue is part of the conserved duplicated region found in catalytically active subunits, and it is postulated that several of these subunits also are truncated by the EDTA treatment. The polypeptides truncated by the EDTA treatment had reduced Ca binding capacities compared to their native subunits, indicating a Ca-binding site within the conserved duplicated region. 63 refs., 7 figs., 2 tabs.
- OSTI ID:
- 508740
- Journal Information:
- Biochemistry (Eaton), Vol. 35, Issue 15; Other Information: PBD: 16 Apr 1996
- Country of Publication:
- United States
- Language:
- English
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