Crystallization of cytoplasmic actin in complex with deoxyribonuclease I.
Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.
D000199:Actins / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D003460:Crystallization; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution; D014961:X-Ray Diffraction
D000199:Actins; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004247:DNA; D003850:Deoxyribonuclease I