Beta-galactosidase: isolation of and antibodies to incomplete chains.
A prematurely terminated polypeptide chain was purified to homogeneity from an Escherichia coli amber mutant strain containing the site of the mutation in the beta-galactosidase structural gene. The polypeptide was highly active against anti-beta-galactosidase, and had an amino acid composition similar to but not identical to that of beta-galactosidase. The molecular weight of the reduced, carboxymethylated chain in 6 m guanidine hydrochloride was found to be 89,000, in excellent agreement with the size predicted from the position of the mutation. This result adds further support to the conclusion that the gene specifies the structure of a single polypeptide chain. Antisera were prepared against partially purified preparations of this polypeptide and a similar one, of molecular weight about 100,000, produced by another amber mutant. These sera had lower titers towards beta-galactosidase than anti-beta-galactosidase. In the double-diffusion test, they reacted towards extracts of nonsense and deletion mutant strains in a pattern similar to that previously observed with anti-beta-galactosidase. A sensitive immunological test for cross-reacting protein was devised based on the inhibition by beta-galactosidase of the reaction between such protein and antibodies prepared against incomplete chains.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000494:pharmacology; D000937:Antigen-Antibody Reactions; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology; D005696:Galactosidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D005827:Genetics, Microbial; D007106:Immune Sera / Q000494:pharmacology; D005779:Immunodiffusion; D008970:Molecular Weight; D009154:Mutation; D010455:Peptides / Q000302:isolation & purification*
D000596:Amino Acids; D000906:Antibodies; D007106:Immune Sera; D010455:Peptides; D005696:Galactosidases