An Amino-terminal Peptide of β-Galactosidase

Abstract

Analysis by the Sanger procedure of cyanogen bromide (BrCN)-treated @-galactosidase and reduced carboxymethylated fi-galactosidase indicated the presence of 23 peptides with 11 different NHt-terminal amino acids.Of two peptides containing NH&erminal threonine, one obtained after gel filtration was found to be threonylhomoserine.Threonylmethionine was shown to be the NHp-terminal sequence of ,&galactosidase by BrCN treatment of the dinitrophenylated protein and isolation of dinitrophenylthreonylhomoserine.Because the dinitrophenyl dipeptide was ether-soluble and could be separated readily, the isolation procedure was applied to crude extracts of Escherichia coli grown in the presence of r4C-methionine or r4C-threonine.Higher levels of radioactivity were found in the dipeptide obtained from a wild type strain induced for fi-galactosidase compared to a noninduced control.Nonsense mutants in which the P-galactosidase chain is expected to be prematurely terminated were tested by the same procedure to obtain evidence for the formation of such chains.For two of these mutants, one which maps close to the operator site and the second which maps near the center of the gene, the same amount of radioactivity was found in the dipeptide from the induced cultures compared to the noninduced.However, for a third mutant, which maps close to the permease gene, clear evidence for higher levels of radioactivity from the induced compared to the noninduced cultures was obtained when growth was carried out at 30' rather than 37'.These results suggest that /3-galactosidase is oriented to its gene in such a manner that the NH2 terminus and the COOH terminus of the protein correspond to the operator and permease ends of the gene, respectively.For genetic correlation, we have been interested in isolation and structure determination of the terminal sequences of /3-*Supported in part by Grants AI-04181 and GM 364 from the National Institutes of Health.