Abstract
Rabbit antiserum raised against pure human hepatic cystathionine beta-synthase was used to precipitate synthase from extracts of radiolabeled cultured fibroblasts derived from 17 homocystinuric patients and two controls. Size analysis of the immunoprecipitates by SDS/polyacrylamide gel electrophoresis revealed that 15 of the 17 synthase-deficient lines synthesized synthase subunits indistinguishable in size from the control (Mr = 63,000). One mutant fibroblast line, previously shown to lack catalytic activity and antigenically cross-reacting material, contained no immunoprecipitable product. Analyses of immunoprecipitated polypeptides synthesized in vitro by cell-free translation of mRNAs prepared from selected mutants confirmed and extended the results from cell extracts. This experimental approach also allowed us to determine the biochemical and genetic defect in a patient with barely detectable synthase subunits in cell extracts. His cultured fibroblasts and those of his father contained two mRNA species, separable by size, coding for equal amounts of two immunoprecipitable polypeptides: one of normal size (Mr = 63,000); the other approximately 7,000 daltons smaller (Mr = 56,000). His mother's fibroblasts made only the Mr = 63,000 species. We conclude that this patient is a compound heterozygote, and that one of his mutant alleles results in the synthesis of a synthase polypeptide missing about 60 amino acid residues.
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