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The effect of hexadecaprenol on molecular organisation and transport properties of model membranes.

100%

Janas T., Nowotarski K., Gruszecki W. I., Janas T.

2000

vol. 47

issue 3

661-673

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure- molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

76%

Olejnik K., Płochocka D., Grynberg M., Goch G., Gruszecki W. I., Basińska T., Kraszewska E.

issue 2

291-300

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function.

Zeaxanthin epoxidation - an in vitro approach

76%

Kuczyńska P., Latowski D., Niczyporuk S., Olchawa-Pajor M., Jahns P., Gruszecki W. I., Strzałka K.

2012

vol. 59

issue 1

105-107

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.

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3 Acta Biochimica Polonica

1 2012

1 2009

1 2000

3 Gruszecki W. I.

1 Basińska T.

1 Goch G.

1 Grynberg M.

1 Jahns P.

1 Janas T.

1 Kraszewska E.

1 Kuczyńska P.

1 Latowski D.

1 Niczyporuk S.

1 Nowotarski K.

1 Olchawa-Pajor M.

1 Olejnik K.

1 Płochocka D.

1 Strzałka K.

Search results

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes.

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

Zeaxanthin epoxidation - an in vitro approach