Effect of sphingomyelin and its metabolites on the activity of human recombinant PLC δ1

Abstract

In an attempt to obtain sufficient quantities of pure phospholipase C δ1 (PLC δ1) necessary for structural and kinetic studies, human fibroblast PLC δ1 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6xHis) fusion protein and purified to homogeneity. From 1 l of E. coli culture 21 mg of pure PLC δ1 was obtained by a two-step purification procedure, which includes Ni 2+-NAT agarose and Mono S cation exchange chromatography. Catalytic properties of recombinant PLC δ1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC δ1 purified from rat liver. Calcium activation of PLC δ1 was dependent on the presence of spermine. Half-maximal activity was attained at 250 and 170 nM of free Ca 2+ in the presence and absence of spermine, respectively. Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP 2. Ceramide inhibits PLC δ1 very weakly. GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC δ1. In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC δ1. The results indicate that the effect of sphingomyelin and its metabolites on PLC δ1 activity depends on the presence of spermine. It is postulated that, among other factors, in vivo, activity of PLC δ1 may depend on the turnover of sphingomyelin.