Ewa Rogalska

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).

In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Differentiating Oxicam Nonsteroidal Anti-Inflammatory Drugs in Phosphoglyceride Monolayers

Meloxicam, piroxicam, and tenoxicam belong to a highly potent oxicam group of nonsteroidal anti-inflammatory drugs. Whereas the structurally similar oxicams have different pharmacokinetics, treatment efficiency, and adverse effects, their common mechanism of action is the inhibition of a membrane enzyme, cyclooxygenase. Because the prerequisite for accessing the cyclooxygenase by the drugs is interaction with the membrane, the focus of the current study was a comparison of how meloxicam, piroxicam, and tenoxicam interact with lipid monolayers used as models of biological membranes. The monolayers were formed with 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection adsorption spectra, and Brewster angle microscopy. The three oxicams are differentiated in the monolayers; meloxicam shows the highest ability to modify membrane fluidity and surface potential, followed by piroxicam and tenoxicam. The dissimilarity of the biological activity of the oxicams may be linked to different interaction with the membrane, as revealed by the present study.

Membrane activity of tetra-p-guanidinoethylcalix[4]arene as a possible reason for its antibacterial properties.

Tetra-p-guanidinoethylcalix[4]arene trifluoroacetate salt (CX1) was synthesized recently as an antibacterial agent. It showed to be active in vitro against various Gram-positive and Gram-negative bacteria. To get more insight in the mechanism of the biological activity of this derivative, it was studied upon interactions with model lipid membranes. Langmuir monolayers were formed with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, and with anionic 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine. The two classes of lipids were used, respectively, as model lipids of the eukaryotic and bacterial cell membranes. The monolayers were exposed to CX1 at different concentrations around the minimum inhibitory concentration found for E. coli . The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy, were employed to study the monolayers. The results obtained show a higher affinity of CX1 for the anionic lipids, indicating importance of charge-charge interactions. On the basis of a comparative study of the behavior of CX1 and that of p-guanidinoethylphenol trifluoroacetate salt, we propose that interplay of charge-charge and apolar interactions between CX1 and lipids is responsible for the important reorganization of model membranes. This proposal may be helpful in developing new antibacterial calixarene derivatives.

The Kinetics, Specificities and Structural Features of Lipases

The four main classes of biological substances are carbohydrates, proteins, nucleic acids and lipids. The first three of these substances have been clearly defined on the basis of their structural features, whereas the property which is common to all lipids is a physicochemical one. Lipids are in fact a group of structurally heterogeneous molecules which are all insoluble in water but soluble in apolar and slightly polar solvents such as ether, chloroform and benzene.

Meloxicam and Meloxicam-β-Cyclodextrin Complex in Model Membranes: Effects on the Properties and Enzymatic Lipolysis of Phospholipid Monolayers in Relation to Anti-inflammatory Activity

Meloxicam, a nonsteroidal anti-inflammatory drug (NSAID), is known as a selective cyclooxygenase-2 inhibitor. Cyclooxygenase-2 is a membrane protein, functionally coupled to an interfacial enzyme, phospholipase A2. Consequently, it may be supposed that the interactions of NSAIDs with lipid membranes play a role in the anti-inflammatory process. In order to investigate the mechanism of this process, Langmuir films formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, or 1,2-myristoyl-sn-glycero-3-phosphoethanolamine were exposed to meloxicam and its beta-cyclodextrin inclusion complex. The monolayers were studied by measuring surface pressure, electric surface potential, Brewster angle micrographs, polarization-modulation infrared reflection-absorption spectra, and phospholipase A2 activity; the inclusion complex was studied using molecular modeling. The results obtained show that the monolayers formed in the presence of meloxicam and its complex are expanded and more liquid-like compared to pure lipids. Both compounds modify hydration of the lipid polar heads, orientation of the molecules, morphology of the domains, and the rate of lipolysis catalyzed by phospholipase A2. The latter effect may be involved in the anti-inflammatory activity of meloxicam. Importantly, the effects observed with the meloxicam-beta-cyclodextrin complex are more pronounced compared to those of the free meloxicam. This observation may be relevant for developing new meloxicam preparations with increased bioavailability.

Fluorinated and hydrogenated cubic phases as matrices for immobilisation of cholesterol oxidase on electrodes

Liquid crystal cubic phases prepared from two different surfactants, monoolein and a fluorinated derivative of an ethoxylated alcohol, have been used as matrices to immobilise the cholesterol oxidase from Rhodococcus sp. The structures of the reverse bicontinuous cubic phases containing pure water or enzyme solution were determined using small angle X-ray scattering (SAXS). Gold and glassy carbon electrodes were modified with the liquid crystals containing the enzyme, and used as sensors for cholesterol detection. The electrochemical response of the glassy carbon/fluorinated system was shown to be a linear function of the cholesterol concentration. It is concluded that the cholesterol oxidase immobilised in an appropriate cubic phase can be successfully used as a sensing element for electrochemical detection of cholesterol in its micellar solutions.

Effects of gemini amphiphilic pseudopeptides on model lipid membranes: A Langmuir monolayer study

Monolayers formed with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] at the air/water interface were used as model membranes for studying a potential biological activity of four newly synthesized gemini amphiphilic pseudopeptides (GAPs); some of the GAPs studied showed interesting self-assembly properties. The capacity of GAPs to self-assemble in different environments let us think that these molecules may find biomedical applications in, e.g., drug delivery or transfection. The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy were used to study monolayers formed with pure GAPs, pure lipids and lipid/GAPs mixtures. The results obtained show that all four GAPs studied can be incorporated in lipid monolayers. The monolayers containing GAPs are expanded and more liquid-like compared to pure lipids. The overall results indicate that the important changes of the properties induced in the model membranes by GAPs are related to their intrinsic conformational flexibility. This feature of GAPs can be easily adjusted by engineering the structure of the spacer present in the polar head, with the aim to modify lipid membranes in a controlled way.

The Mechanism of Metal Cation Binding in Two Nalidixate Calixarene Conjugates. A Langmuir Film and Molecular Modeling Study

The two new p-tert-butylcalix[4]arene derivatives described here bear one or two nalidixic acid arms linked to the lower calixarene rim via the quinolone carboxylate moiety. These derivatives were synthesized in order to investigate two important features of molecules conceived as potential antibiotics, namely, metal cation complexation and interfacial properties, and the way in which they interrelate. The properties of the calixarene derivatives were studied in monomolecular films spread on pure water and on aqueous subphases containing biologically relevant mono- and divalent metal cations. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection absorption spectroscopy, and molecular modeling. Molecular modeling shows that important differences exist, first, between the structure and stability of the complexes formed with the two derivatives and, second, between their mono- and dication complexes. Correlating the properties of the monolayers with those of the modeled molecules lets us propose that the derivatives bearing one or two nalidixic pending arms form preferentially inter- and intramolecular complexes, respectively. The results obtained in this study indicate that a possible biological role of the nalidixic arms grafted on the calixarene crown may be revealed upon cation complexation.

Interfacial approach to polyaromatic hydrocarbon toxicity: phosphoglyceride and cholesterol monolayer response to phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene.

Interactions of phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene (polyaromatic hydrocarbons) with model phospholipid membranes were probed using the Langmuir technique. The lipid monolayers were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and cholesterol. Surface pressure and electrical surface potential were measured on mixed phospholipid/PAH monolayers spread on a pure water subphase. The morphology of the mixed monolayers was followed with Brewster angle microscopy. Polarization-modulation infrared reflection-absorption spectroscopy spectra obtained on DPPE/benzo[a]pyrene showed that the latter interacts with the carbonyl groups of the phospholipid. On the other hand, the activity of phospholipase A2 toward DLPC used as a probe to locate benzo[a]pyrene in the monolayers indicates that the polyaromatic hydrocarbons are not accessible to the enzyme. The results obtained show that all PAHs studied affect the properties of the pure lipid, albeit in different ways. The most notable effects, namely, film fluidization and morphology changes, were observed with benzo[a]pyrene. In contrast, the complexity of mixed lipid monolayers makes the effect of PAHs difficult to detect. It can be assumed that the differences observed between PAHs in monolayers correlate with their toxicity.