Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.
The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.
Horstman
D A
DA
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
DeStefano
K
K
Carpenter
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D006639:Histidine; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008322:Mammals; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051966:Phospholipase C gamma; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D018411:Spodoptera; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D046911:Macromolecular Substances; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010738:Type C Phospholipases; D051966:Phospholipase C gamma