ALKALINE PHOSPHATASE AND 5'-NUCLEOTIDE PHOSPHODIESTERASE FROM BOVINE INTESTINE: STRUCTURAL RELATIONSHIP AS DETERMINED BY ANTIBODIES AND ACTIVE SITE AMINO ACID SEQUENCES
Abstract
Alkaline phosphatase (ALPase) and 5'-nucleotide phosphodiesterase (PDase) from bovine intestine have many similar structural and catalytic properties. Amino acid sequence analysis of active site sequences and antibody recognition were used to establish the extent of the structural relationship between the two enzymes. Antibodies to native ALPase and antibodies to native PDase cross-reacted with PDase and ALPase, respectively. These antibodies cross-reacted with denatured enzymes but not with enzymes whose carbohydrate had been modified or removed. These results suggest that the antibodies to native enzymes are directed against the carbohydrate moieties of the enzymes. This conclusion is supported by the very similar carbohydrate composition of ALPase and PDase. Antibodies to denatured PDase cross-react with ALPase when the carbohydrate structures of ALPase are altered or removed. This antibody does not bind to native ALPase or PDase. This suggests that antibodies to denatured PDase recognize amino acid sequence similarities buried inside the native enzymes. The radioactive covalent intermediates of PDase and ALPase were isolated during incubation with 5'-(('32)P)-thymidine triphosphate and (('32)P)-inorganic phosphate, respectively. ALPase and PDase radioactive cyanogen bromide peptides of approximately 60 amino acids were purified and sequenced. The active site residue of bovine ALPase is serine and the sequence surrounding the serine is very similar to the sequence surrounding the active site serine of E. coli ALPase. There are limited amino acid sequence similarities between the CNBr active site peptides of ALPase and PDase. However, the PDase active site residue is threonine and the threonine is not located in a region of similarity. We are not aware of any other reports of threonine active site residues that form a covalent intermediate.
Degree
Ph.D.
Subject Area
Biochemistry
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