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We investigated the biosynthesis of cystathionine beta-synthase (EC 4.2.1.22) in a cell-free translation system programmed with rat liver mRNA and in slices of rat liver. The enzyme was recovered by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only a single mRNA species, coding for a 63,000-dalton polypeptide, was detected when rat liver mRNA was assayed by cell-free translation. On the other hand, two polypeptides were recovered by immunoprecipitation from fresh liver extracts: a predominant Mr = 63,000 polypeptide and a minor Mr = 48,000 polypeptide. When such extracts were incubated at 4 degrees C for 7 days, the synthase activity increased 2-3-fold with a concomitant disappearance of the Mr = 63,000 polypeptide and some increase of the Mr = 48,000 polypeptide. Moreover, the specific activity of synthase containing the smaller subunits was now found to be approximately 60-fold higher than that containing the larger ones. At least in part, this increased specific activity reflected a 30-fold greater affinity for homocysteine. The changes in subunit size and activity could be prevented in vitro by protease inhibitors such as N alpha-p-tosyl-L-lysine chloromethyl ketone, antipain, and leupeptin, but not by several other protease inhibitors. Pulse-chase experiments with slices of rat liver suggested a slow, post-translational conversion of the Mr = 63,000 polypeptide to the Mr = 48,000 polypeptide. Taken together, our findings are consistent with the possibility that the large subunit form of synthase is essentially inactive under physiologic conditions, and that synthase activity is regulated by limited proteolysis.

A single specific radiolabeled polypeptide with an apparent Mr = 63,000 was recovered when cystathionine beta-synthase (EC 4.2.1.22) was precipitated from extracts of radiolabeled cultured human fibroblasts with an antiserum raised against pure human liver synthase, and the immunocomplexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolysis of this fibroblast subunit and of the subunit of pure human liver synthase (Mr = 48,000) produced similar peptide patterns. Pulse-chase experiments, however, did not provide any evidence for post-translational modification of the fibroblast synthase subunit into a smaller “hepatic” form. Immunoprecipitation of polypeptides synthesized in vitro from human fibroblast mRNA revealed a polypeptide with the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the synthase subunit found in whole cell extracts. We conclude that the Mr = 63,000 subunit is the primary translational product of the gene for cystathionine beta-synthase in human fibroblasts.