PURIFICATION AND CHARACTERIZATION OF THE GLYOXYSOMAL ENZYME MALATE SYNTHASE FOLLOWING SEED-GERMINATION IN PINUS-TAEDA

Malate synthase (EC 4.1.3.2) has been purified to homogeneity from ger minated loblolly pine (Pinus taeda L.) megagametophytes and polyclonal antibodies elicited in rabbits. The enzyme is an octamer with a nativ e molecular mass of 520,000 kDa and a subunit molecular mass of 62,000 kDa. Loblolly pine malate synthase is not a glycoprotein. The enzyme does not react with Schiffs reagent, bind concanavalin A-Sepharose, or deglycosylated with N-glycosidase F. In vivo and in vitro synthesized malate synthase have identical molecular masses to that of the purifi ed protein. Cell-free activity assays and immunoblots demonstrate that the enzyme is developmentally regulated in loblolly pine seeds. Concu rrent with the initial rates of lipid catabolism, malate synthase acti vity and steady-state protein were very low in the mature seed megagam etophyte, increased dramatically following germination, peaking at aro und 10 days after imbibition, then subsequently declined with tissue s enescence. In the embryo, malate synthase activity and protein levels were undetectable in the mature and stratified seed, but present at lo w levels at radicle emergence. To our knowledge, this study represents the first report of an isolation, immunological characterization, and developmental regulation of malate synthase from a gymnosperm source.