On the importance of anandamide structural features for its interactions with DPPC bilayers: effects on PLA2 activity

The acylethanolamide anandamide (AEA) occurs in a variety of mammalian tissues and, as a result of its action on cannabinoid receptors, exhibits several cannabimimetic activities. Moreover, some of its effects are mediated through interaction with an ion channel-type vanilloid receptor. However, the chemical features of AEA suggest that some of its biological effects could be related to physical interactions with the lipidic part of the membrane. The present work studies the effect of AEA-induced structural modifications of the dipalmitoylphosphatidylcholine (DPPC) bilayer on phospholipase A2 (PLA2) activity, which is strictly dependent on lipid bilayer features. This study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene fluorescence, demonstrates that the effect of AEA on PLA2 activity is concentration-dependent. In fact, at low AEA/DPPC molar ratios (from R = 0.001 to R = 0.04), there is an increase of the enzymatic activity, which is completely inhibited for R = 0.1. X-ray diffraction data indicate that the AEA affects DPPC membrane structural properties in a concentration-dependent manner. Because the biphasic effect of increasing AEA concentrations on PLA2 activity is related to the induced modifications of membrane bilayer structural properties, we suggest that AEA-phospholipid interactions may be important to produce, at least in part, some of the similarly biphasic responses of some physiological activities to increasing concentrations of AEA.     

Modifications induced by plasma of gestational hypertensive women on the Na+/K+-ATPase obtained from human placenta

Pyrimidines and purine (deoxy)nucleotides are the building blocks of DNA and RNA. Nucleoside diphosphate sugars, e.g. UDP-glucose, are the reactive intermediates in the synthesis of nearly all glycosidic bonds between sugars.In mammals the requirement for pyrimidines is met by UMP de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The exceptional compartmentation of the de novo synthesis with respect to mitochondrially-bound dihydroorotate dehydrogenase ('DHOdehase' or 'DHODH', EC 1.3.99.11) is one focus of the present work. DHODH activity was determined by the dihydroorotate-dependent oxygen consumption or by the UV absorption of the product orotate with mitochondria isolated from rodent and porcine tissues. For comparison, the cytochrome c and choline-dependent oxygen consumption of mitochondria from different tissues was measured. The highest specific activity of the rat DHODH was found in liver (2.3 × 10-3 µmol/min × mg protein) > kidney > heart. The application of known enzyme inhibitors Brequinar Sodium and Leflunomide for DHODH and sodium cyanide for cytochrome c oxidase verified the specificity of the activity tests used. The relation of DHODH activity versus that of cytochrome c oxidase revealed the lowest ratios in heart mitochondria and the highest in liver mitochondria. Since disorders in the mitochondrial energy metabolism could entail severe impairment of pyrimidine biosynthesis via respiratory-chain coupled DHODH, it is suggested to include improvement of pyrimidine nucleotide status in therapy protocols. (Mol Cell Biochem 174: 125–129, 1997)     

Different modulation of phospholipase A2 activity by saturated and monounsaturated N-acylethanolamines

The physiological functions of N-acylethanolamines (NAEs) are poorly understood, although many functions were suggested for these naturally occurring membrane components of plants and animals. The binding with cannabinoid receptors CB1 and CB2 was demonstrated for some NAEs, such as anandamide. However, the chemical nature of these molecules suggests that some of their biological effects on biomembranes could be related, at least partially, to physical interactions with the lipid bilayer. The present work studies the effect of saturated and monounsaturated NAEs on phospholipase A2 (PLA2) activity, which is dependent on lipid bilayer features. The present study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) fluorescence, demonstrates that the acyl chain length and the presence of a single double bond are crucial for the enzymatic activity modulation by NAEs. In fact, saturated NAEs with 10 carbon atoms don't affect the PLA2 activity, while NAEs with 12 and 16 carbon atoms largely activate the enzyme. On the other hand, an acyl chain length of 18 carbon atoms, with or without the presence of a double bond, only slightly affects the enzymatic activity. A structural model for NAE-lipid interactions is proposed in order to explain the differences in PLA2 activity modulation by these fatty acid derivatives.     

Anandamide and its congeners inhibit human plasma butyrylcholinesterase. Possible new roles for these endocannabinoids?

Butyrylcholinesterase (BChE), a serine hydrolase biochemically related to the cholinergic enzyme Acetylcholinesterase (AChE), is found in many mammalian tissues, such as serum and central nervous system, but its physiological role is still unclear. BChE is an important human plasma esterase, where it has detoxifying roles. Furthermore, recent studies suggest that brain BChE can have a role in Alzheimer’s disease (AD). The endocannabinoid arachidonoylethanolamide (anandamide) and other acylethanolamides (NAEs) are almost ubiquitary molecules and are physiologically present in many tissues, including blood and brain, where they show neuroprotective and anti-inflammatory properties. This paper demonstrates that they are uncompetitive (oleoylethanolamide and palmitoylethanolamide) or non competitive (anandamide) inhibitors of BChE (Ki in the range 1.32-7.48 nM). On the contrary, NAEs are ineffective on AChE kinetic features. On the basis of the X-ray crystallographic structure of human BChE, and by using flexible docking procedures, an hypothesis on the NAE-BChE interaction is formulated by molecular modeling studies. Our results suggest that anandamide and the other acylethanolamides studied could have a role in the modulation of the physiological actions of BChE.     

Temperature-induced molten globule-like state in human alpha1-acid glycoprotein: an infrared spectroscopic study

Despite extensive investigations on thermal denaturation of alpha(1)-acid glycoprotein (AGP) using a variety of techniques, structural features of the folded-unfolded state in terms of residual secondary structures and the structural transitions involved in this process have not been fully characterized. In this study we employed FT-IR spectroscopy to investigate the thermal unfolding and reversibility of temperature-induced changes in AGP. The data revealed a fully reversible beta-sheet-rich protein which exhibits a molten globule-like state, an important protein folding intermediate. 2D-IR COS revealed the sequence of the conformational changes occurring before denaturation and confirmed the formation of this intermediate which was further supported by CD spectroscopy. On account of the similarities in the FT-IR spectra of AGP with those of porcine odorant-binding protein (OBP), homology modeling of AGP using OBP as template was performed. The resemblance of AGP and OBP 3D structures confirmed the similarities of data obtained using FT-IR spectroscopy. Overall, FT-IR spectroscopy appears to be useful for investigating the structural characteristics and stability of proteins whose 3D structures are unavailable and for assessing the molten globule-like state in small beta-sheet-rich proteins.     

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines

The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA or Trp-212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp-134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue.     

Interaction of the herbicide atrazine with model membranes. II: Effect of atrazine on fusion of phospholipid vesicles

The effect of atrazine on Ca2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb3+/dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca2(+)-induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca2+. It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca2(+)-induced fusion of vesicles is due to a different localization of the pesticide in the membrane.     

S-100b protein regulates aggregation and fusion of cardiolipin vesicles

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.