Immunoreactive protein in adenosine deaminase deficient human lymphoblast cell lines.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was examined in human lymphoblast cell lines from normal and adenosine deaminase-deficient individuals as well as individuals heterozygous for adenosine deaminase deficiency. Adenosine deaminase activity was determined by a specific enzymatic assay and compared to immunoreactive adenosine deaminase protein (or cross-reacting material) determined by radioimmunoassay, in order to investigate mutations affecting adenosine deaminase. Two different antisera, raised in goat and rabbit against human adenosine deaminase, had different sensitivities and apparent specificities when used for radioimmunoassay. Rabbit antisera provided the most sensitive assay of normal enzyme, whereas goat antiserum provided the most sensitive assay for detection of mutant proteins. A wide range of values for the ratio of immunoreactive protein to activity was observed for the adenosine deaminase deficient cell lines, ranging from near normal to 23 times normal. The cell lines with high ratios appear to contain large amounts of catalytically defective or inactive protein. The amount of mutant protein detected by radioimmunoassay in the deficient cell lines depends upon the antiserum utilized, making as much as a 50-fold difference. The heterozygous lines contain approximately half of the normal amounts of immunoreactive protein and activity, and thus have a normal ratio of the two. Two cell lines partially deficient in adenosine deaminase appear to contain large amounts of an unstable adenosine deaminase protein with partially impaired activity. Immunoreactive protein was visualized in extracts from several cell lines, after electrophoresis and transfer to activated paper, by labeling with immunological probes and autoradiography.