scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure

1978 ◽  
Vol 175 (1) ◽  
pp. 115-124 ◽  
Author(s):  
D T Pechey ◽  
A B Graham ◽  
G C Wood
Keyword(s):  
Guinea Pig ◽  
Phospholipid Bilayer ◽  
Permeability Barrier ◽  
Uridine Diphosphate ◽  
Membrane Bilayer ◽  
Arrhenius Plots ◽  
Acceptor Activity ◽  
Microsomal Membranes ◽  
Acyl Chains ◽  
Phospholipid Acyl Chains

Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a ‘phase separation’ or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.

scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity (reverse reaction)

1980 ◽  
Vol 185 (2) ◽  
pp. 521-526 ◽  
Author(s):  
J Cummings ◽  
A B Graham ◽  
G C Wood
Keyword(s):  
Guinea Pig ◽  
Transition Point ◽  
Reverse Reaction ◽  
Phospholipid Bilayer ◽  
Bilayer Structure ◽  
Uridine Diphosphate ◽  
Microsomal Enzyme ◽  
Arrhenius Plots ◽  
Microsomal Membranes ◽  
Donor Activity

Arrhenius plots of the non-latent UDP-glucuronlytransferase reverse reaction (p-nitrophenyl glucuronide donor) activity of guinea-pig microsomal membranes prepared with 15 mM-KCl were linear from 5 to 40 degrees C. These plots for other preparations from guinea-pig and rat liver (i.e. preparations that show transferase latency) exhibited two linear regions intersecting at a transition point near 19-21 degrees C. This discontinuity was abolished when latency was removed by treating the membranes with perturbants of phospholipid-bilayer structure. Thus the temperature-depdendnces of the reverse reaction catalysed by the enzymes of these various preparations are similar to those of the corresponding forward reactions [Pechey, Graham & Wood (1978) Biochem. J. 175, 115-1124]. Perturbants activated the enzyme of KCl-prepared guinea-pig microsomal membranes only slightly and caused no significant alteration to Arrhenius plots of its forward or reverse reaction activities. These results support the ‘compartmentation’ theory of UDP-glucuronyltransferase lactency.

scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

1977 ◽  
Vol 163 (1) ◽  
pp. 117-124 ◽  
Author(s):  
A B Graham ◽  
D T Pechey ◽  
K C Toogood ◽  
S B Thomas ◽  
G C Wood
Keyword(s):  
Guinea Pig ◽  
Degradation Products ◽  
Active Form ◽  
Gel Filtration ◽  
Transferase Activity ◽  
Uridine Diphosphate ◽  
Pig Liver ◽  
Highly Active ◽  
Microsomal Membranes ◽  
Guinea Pig Liver

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.

How do mechanosensitive channels sense membrane tension?

2016 ◽  
Vol 44 (4) ◽  
pp. 1019-1025 ◽  
Author(s):  
Tim Rasmussen
Keyword(s):  
Membrane Lipids ◽  
Osmotic Shock ◽  
Md Simulations ◽  
Fatty Acyl ◽  
Membrane Bilayer ◽  
Cross Sectional ◽  
Conducting Path ◽  
Lipid Chain ◽  
Acyl Chains

Mechanosensitive (MS) channels provide protection against hypo-osmotic shock in bacteria whereas eukaryotic MS channels fulfil a multitude of important functions beside osmoregulation. Interactions with the membrane lipids are responsible for the sensing of mechanical force for most known MS channels. It emerged recently that not only prokaryotic, but also eukaryotic, MS channels are able to directly sense the tension in the membrane bilayer without any additional cofactor. If the membrane is solely viewed as a continuous medium with specific anisotropic physical properties, the sensitivity towards tension changes can be explained as result of the hydrophobic coupling between membrane and transmembrane (TM) regions of the channel. The increased cross-sectional area of the MS channel in the active conformation and elastic deformations of the membrane close to the channel have been described as important factors. However, recent studies suggest that molecular interactions of lipids with the channels could play an important role in mechanosensation. Pockets in between TM helices were identified in the MS channel of small conductance (MscS) and YnaI that are filled with lipids. Less lipids are present in the open state of MscS than the closed according to MD simulations. Thus it was suggested that exclusion of lipid fatty acyl chains from these pockets, as a consequence of increased tension, would trigger gating. Similarly, in the eukaryotic MS channel TRAAK it was found that a lipid chain blocks the conducting path in the closed state. The role of these specific lipid interactions in mechanosensation are highlighted in this review.

scholarly journals Differing Membrane Interactions of Two Highly Similar Drug-Metabolizing Cytochrome P450 Isoforms: CYP 2C9 and CYP 2C19

2019 ◽  
Vol 20 (18) ◽  
pp. 4328 ◽  
Author(s):  
Ghulam Mustafa ◽  
Prajwal P. Nandekar ◽  
Neil J. Bruce ◽  
Rebecca C. Wade
Keyword(s):  
Cytochrome P450 ◽  
Transmembrane Helix ◽  
Phospholipid Bilayer ◽  
Endoplasmic Reticulum Membrane ◽  
Globular Domain ◽  
Membrane Interactions ◽  
Membrane Bilayer ◽  
Similar Drug ◽  
Human Cytochrome ◽  
High Sequence Conservation

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

scholarly journals Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa

1976 ◽  
Vol 155 (3) ◽  
pp. 607-613 ◽  
Author(s):  
P R Flanagan ◽  
S H Zbarsky
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Large Loss ◽  
Arrhenius Plots ◽  
The Poor ◽  
Km Value ◽  
Soluble Material

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 × 10(-5) M with thymidine 3′-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.

Influence of the sterol aliphatic side chain on membrane properties: a molecular dynamics study

2015 ◽  
Vol 17 (35) ◽  
pp. 22736-22748 ◽  
Author(s):  
João R. Robalo ◽  
J. P. Prates Ramalho ◽  
Daniel Huster ◽  
Luís M. S. Loura
Keyword(s):  
Molecular Dynamics ◽  
Membrane Properties ◽  
Side Chain ◽  
Side Chains ◽  
Aliphatic Side Chain ◽  
Acyl Chains ◽  
Hydrophobic Matching ◽  
Phospholipid Acyl Chains

Cholesterol provides best hydrophobic matching, induces maximal membrane ordering, and displays highest preference for saturated phospholipid acyl chains, among a homologous ser ies of sterols with side chains of varying lengths.

scholarly journals Vertical fluctuations of phospholipid acyl chains in bilayers

FEBS Letters ◽  
1987 ◽  
Vol 223 (1) ◽  
pp. 20-24 ◽  
Author(s):  
John R. Wardlaw ◽  
William H. Sawyer ◽  
Kenneth P. Ghiggino
Keyword(s):  
Acyl Chains ◽  
Phospholipid Acyl Chains

scholarly journals COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1971 ◽  
Vol 49 (1) ◽  
pp. 150-158 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Electron Transport ◽  
Guinea Pig ◽  
Enzyme Activities ◽  
Transport Systems ◽  
Zymogen Granule ◽  
Electron Transport Chains ◽  
Microsomal Membranes ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Secretory Products

A comparative study of the enzymic activities of membrane fractions derived from guinea pig pancreatic homogenates has yielded the following results: Rough microsomal membranes (derived from the rough ER) have the reductase activities of the two microsomal electron transport systems but lack enzyme activities of Golgi-type (TPPase) and plasmalemmal-type (5'-nucleotidase, ß-leucyl naphthylamidase, Mg-ATPase). Smooth microsomal membranes (derived primarily from the Golgi complex), zymogen granule membranes, and plasmalemmal fractions possess overlapping enzyme activities of plasmalemmal type, in different relative concentrations for each fraction. In addition, the smooth microsomal membranes exhibit TPPase and ADPase activity and share with rough microsomes the reductase activities of the two electron transport chains. Taken together with recent data on the lipid composition of the same fractions (2), these results indicate that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and hence do not mix with one another during the transport of secretory products.

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