Year: 1969
Type: article
Abstract: Two inactive fragments of staphylococcal nuclease (a protein consisting of 149 residues, devoid of sulfhydryl groups and disulfide bonds) have been prepared; one (nuclease-P(,126)) contains Residues 1 through 126, and the other (nuclease-P(127_ 49)) contains Residues 127 through 149.Studies of nuclease-P(I 1 26), employing circular dichroism, optical rotation, immunodiffusion, and solvent perturbation, reveal a loose and disorganized structure very different from that of nuclease.Nuclease-P( 127 .149) is also structureless, as determined by measurements of circular dichroism and the fluorescence spectrum of the tryptophan residue.It was shown previously that the cleavage of nuclease by trypsin in the presence of thymidine 3',5'-diphosphate and calcium ions yields nuclease-P(6.4 9 ) (Residues 6 through 149), which has full enzymic activity and a conformation similar to that of nuclease.An enzymically active (8 to 10% of the native level) derivative, nuclease-T, composed of two fragments bonded noncovalently, nuclease-T-P(6 48) (Residues 6 to 48) and nuclease-T-P( 49 .EO-149) (mixture of Residues 49 through 149 and 50 through 149), is subsequently formed.The association of separated nuclease-T-P(648) and -P(49, 50-149) regenerates both enzymic activity (of nuclease-T) and a three-dimensional structure similar to that of nuclease.When the fragments are mixed, noncovalent bonding of nuclease-P( 1 _2 6 ) with nuclease-T-P( 49 , 50-149), but not with nuclease-P(2 7 ?.49), is indicated by generation of enzymic activity, increase in ellipticity (from 215 through 240 m~a), positive reaction with antinuclease serum, and a shift in the emission maximum of fluorescence of the single tryptophan residue.Trypsin digestion of noncovalently bonded nuclease-P(,-1 2 6 ) and nuclease-T-P(4 9 , 5-149) in the presence of thymidine 3',5'-diphosphate and calcium ions yields nuclease-T and small fragments derived from Residues 1 through 5 and 49 through 126.Thus, nuclease-T-P 49.6-149) appears to bind only with the nuclease-T-P(6 4 8 ) portion of nuclease-P(-1 2 6 ) (Residues 6 through 48) to form a conformation resembling that of nuclease-T, whereas the other portions of the polypeptide chain of nuclease-P(. 1 2 6 ) protrude from the nuclease-T structure as essentially random coils.* A preliminary account of this work was presented at the 53rd annual meeting of the Federation of American Societies for Experimental Biology (1).
Source: Journal of Biological Chemistry
Authors: H Taniuchi, C B Anfinsen
Institution: National Institutes of Health
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1569300949