ATP: citrate lyase of Rhodotorula gracilis: purification and properties

Abstract

ATP: citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520 000 and consisted of four identical subunits ( M r = 120 000). Two minor low molecular weight bands were observed on SDS-PAGE ( M r 51 000 and M r 49 000). Trypsin digestion experiments indicated that these could have been the result of limited proteolysis by an endogenous trypsin-like proteinase . In this respect, it resembles the mammalian ATP:citrate lyase. The enzyme was stimulated by NH 4 + ions and inhibited by palmitoyl, lauroyl, oleoyl, myristoyl and stearoyl-CoA esters, glutamate and glucose 6-phosphate but not by acetyl-CoA or shorter chain fatty acyl-CoA esters. The enzyme exhibited normal Michaelis-Menten kinetics for citrate; however there was a 3-fold increase in K m with a high concentration of Cl − ions (0.25 M). The possible regulatory roles of ATP:citrate lyase in R. gracilis are discussed in the light of these findings.