The molecular pathology of the porphobilinogen (PBG)-deaminase deficiency in heterozygotes for acute intermittent porphyria (AIP) was investigated by means of biochemical and immunologic techniques. The stable enzyme-substrate intermediates (A, B, C, D, and E) of PBG-deaminase were separated by anion-exchange chromatography of erythrocyte lysates from heterozygotes for AIP and normal individuals. In normal lysates, the intermediates eluted in a characteristic pattern with decreasing amounts of activity (A > B > C > D > E), the combined A and B intermediates representing >75% of total recovered activity. In contrast, two different profiles were observed in lysates from heterozygotes for AIP. In most heterozygotes, the elution profile was similar to that of normal individuals, but each intermediate was reduced ~50%. A second profile in which the C intermediate had disproportionately higher activity than the A or B intermediates was observed in asymptomatic heterozygotes with high urinary levels of PBG (>5 μg/ml) as well as in heterozygotes during acute attacks. These findings suggested that the C intermediate (the dipyrrole-enzyme intermediate) may be rate limiting in the stepwise conversion of the monopyrrole, PBG, to the linear tetrapyrrole, hydroxymethylbilane. To investigate further the nature of the enzymatic defect in AIP, sensitive immunotitration and immunoelectrophoretic assays were developed with the aid of a rabbit anti-human PBG-deaminase IgG preparation produced against the homogeneous enzyme. Equal amounts of erythrocyte lysate activity from 32 heterozygotes for AIP from 22 unrelated families and 35 normal individuals were immunoelectrophoresed. There were no detectable differences in the amounts of cross-reactive immunologic material (CRIM) in lysates from the normal individuals and 25 heterozygotes from 21 of the 22 unrelated families with AIP. In contrast, when equal enzymatic activities were coimmunoelectrophoresed, all seven heterozygotes from one family had ~1.6 times the amount of CRIM compared with that detected in normal lysates. Consistent with these findings, immunotitration studies also demonstrated similar quantities of noncatalytic CRIM in lysates from this AIP family. When equal activities of the individual A, B, C, and D enzyme-substrate intermediates from normal and CRIM-positive erythrocytes were immunoelectrophoresed, increased amounts of immunoreactive protein were observed for each intermediate, B > A ≃ C ≃ D, from the CRIM-positive AIP variants. On the basis of these findings, it is hypothesized that the enzymatic defect in the CRIM-postivie AIP family resulted from a mutation in the structural gene for PBG-deaminase which altered the catalytic as well as a substrate binding site. These studies of the enzymatic defect provide the first demonstration of genetic heterogeneity in AIP.
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