Effect of sphingomyelin and its metabolites on the activity of human recombinant PLC delta 1.
In an attempt to obtain sufficient quantities of pure phospholipase C delta 1 (PLC delta 1) necessary for structural and kinetic studies, human fibroblast PLC delta 1 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6xHis) fusion protein and purified to homogeneity. From 11 of E. coli culture 21 mg of pure PLC delta 1 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 delta 1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC delta 1 purified from rat liver. Calcium activation of PLC delta 1 was dependent on the presence of spermine. Half-maximal activity was attained at 250 and 170 nM of free Ca2+ in the presence and absence of spermine, respectively. Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP2. Ceramide inhibits PLC delta 1 very weakly. GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC delta 1. In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC delta 1. The results indicate that the effect of sphingomyelin and its metabolites on PLC delta 1 activity depends on the presence of spermine. It is postulated that, among other factors, in vivo, activity of PLC delta 1 may depend on the turnover of sphingomyelin.
Matecki
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Department of Clinical Biochemistry, Medical University of Gdansk, Poland.
Stopa
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M
Was
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Pawelczyk
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T
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D010455:Peptides / Q000378:metabolism; D054803:Phospholipase C delta; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism* / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D013109:Sphingomyelins; C033223:polyhistidine; D006639:Histidine; D010738:Type C Phospholipases; C518024:PLCD1 protein, human; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat