Biosynthesis and proteolytic activation of cystathionine beta-synthase in rat liver.
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.
D000818:Animals; D002474:Cell-Free System; D003541:Cystathionine beta-Synthase / Q000096:biosynthesis*; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000096:biosynthesis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D011480:Protease Inhibitors; D014357:Trypsin; D006836:Hydro-Lyases; D003541:Cystathionine beta-Synthase