Expression, purification and kinetic properties of human recombinant phospholipase C delta 3.
To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.
Pawełczyk
T
T
Department of Clinical Biochemistry, Medical University of Gdańsk, Poland. tkpaw@amedec.amg.gda.pl
Matecki
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A
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D054803:Phospholipase C delta; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C518024:PLCD1 protein, human; C518028:PLCD3 protein, human; D054803:Phospholipase C delta