Wyniki wyszukiwania

1

Expression, purification and kinetic properties of human recombinant phospholipase C delta3

100%

Pawelczyk T., Matecki A.

Acta Biochimica Polonica

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1997

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tom 44

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nr 2

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.

2

The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain

80%

Kowara R., Gryckiewicz E., Matecki A., Pawelczyk T.

Acta Biochimica Polonica

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1999

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tom 46

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nr 2

Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-γ was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UV spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UV spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 4-phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. These data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at least overlapping. The site of PIP and PIP2 interaction with PKC-γ is distinct from that of phorbol ester binding site.

3

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Identification of adenosine receptors in basolateral membranes of rat renal cortex

61%

Jakubowski Z., Skowronski R., Matecki A., Mohuczy D., Pawelczyk Z., Stepinski J., Angielski S.

Acta Physiologica Polonica

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1988

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tom 39

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nr 1

4

Role of carnitine in maintenance of acetyl-CoA level and functional integrity of cholinergic neuroblastoma cells

61%

Bielarczyk H., Cwiowska J., Jankowska A., Tomaszewicz M., Matecki A., Szutowicz A.

Acta Biochimica Polonica

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2003

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tom 50

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nr Supplement 1

Ograniczanie wyników

3 Acta Biochimica Polonica

1 Acta Physiologica Polonica

4 Matecki A.

2 Pawelczyk T.

1 Angielski S.

1 Bielarczyk H.

1 Cwiowska J.

1 Gryckiewicz E.

1 Jakubowski Z.

1 Jankowska A.

1 Kowara R.

1 Mohuczy D.

1 Pawelczyk Z.

1 Skowronski R.

1 Stepinski J.

1 Szutowicz A.

1 Tomaszewicz M.

1 2003

1 1999

1 1997

1 1988

Expression, purification and kinetic properties of human recombinant phospholipase C delta3

The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain

Identification of adenosine receptors in basolateral membranes of rat renal cortex

Role of carnitine in maintenance of acetyl-CoA level and functional integrity of cholinergic neuroblastoma cells