Monomolecular Film Technique Research Articles

Interfacial properties of the polyheme cytochrome c3 superfamily from Desulfovibrio.

In order to compare the interfacial behavior of the polyheme cytochromes c which belong to the cytochrome c3 superfamily, the monomolecular film technique was used to determine whether and how these metalloproteins interact with (phospho)lipids). Measurements of the variations of surface pressure and surface potential versus time have shown differences in their penetration capacity into phosphatidylcholine, dicaprin, and phosphatidylglycerol films. The Desulfovibrio vulgaris Hildenborough cytochrome with 16 hemes (Hmc) and Desulfovibrio desulfuricans Norway tetra- and octaheme cytochromes c3, which have been assumed to be soluble periplasmic molecules, may be considered as extrinsic membrane proteins, unlike the D. vulgaris Hildenborough cytochrome c3 (Mr 13 000). The interfacial properties are discussed in terms of the available three-dimensional structural data, the electrostatic potential calculation, and the results obtained by hydrophobic cluster analysis of the cytochrome sequences. The very different behavior of the two cytochromes c3 (Mr 13 000) enlightens the role of a particular surface loop in the interaction with a model membrane. A functional interpretation is proposed assuming that the D. vulgaris Hildenborough Hmc and both cytochromes c3 (Mr 13 000) and (Mr 26 000) from the Norway strain might provide the link between periplasmic hydrogen oxidation and cytoplasmic sulfate reduction.

Interfacial Binding of Human Gastric Lipase to Lipid Monolayers, Measured with an ELISA

Two sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin--peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN.m-1 were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mumol/(min.mg of enzyme)]. At a given lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Glyceride synthesis catalyzed by cutinase using the monomolecular film technique.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

Lipase stereoselectivity and regioselectivity toward three isomers of dicaprin: A kinetic study by the monomolecular film technique

AbstractHere we present a kinetic study on the steroselectivity and regioselectivity of 23 purified lipases of animal and microbial origin. This work, concerning a general problem of the mechanism of lipase–substrate molecular recognition, was performed using pure dicaprin isomers: 1,2‐sn‐dicaprin, 2,3‐sn‐dicaprin, and 1,3‐sn‐dicaprin spread as monomolecular films at the air–water interface. The first two isomers are optically active antipodes (enantiomers), forming stable films up to 40 mN m−1, while the last is a prochiral compound, with a surface pressure of collapse of 32 mN m−1. To our knowledge, this is the first report on the use of three diglyceride isomers as lipase substrates under identical and controlled physicochemical conditions. The lipases tested display a typical behaviour, characteristic of each enzyme, which allowed us to classify the lipases in groups according to (1) the profiles of enzyme velocity as a function of surface pressure, (2) their preferences for a given diglyceride isomer, quantified using new parameters termed steroselectivity index (S.I.), vicinity index (V.I.), and surface pressure threshold (S.P.T.). The general observation, true for all the enzymes tested, is that the three substrates are well differentiated, and the differentiation is more pronounced at high interfacial energy (low surface pressure). This observation supports our hypothesis that lipase conformational changes, resulting from the enzymesurface interaction, affect the enzymes' specificities. Generally speaking, the stereopreference for either sn‐1 or sn‐3 position on glycerides is maintained both in the case of di‐ and tri‐glycerides. © 1995 Wiley‐Liss, Inc.

Penetration of phospholipid monolayers by opioid peptides and opiate drugs (agonists and antagonists)

The monomolecular film technique was used to compare the specific interaction of opioid peptides (Leu-enkephalin, Met-enkephalin, dynorphin A and β-endorphin) and opiate agonists (alkaloids such as levorphanol and ethylketocyclazocine) and opiate antagonists (naloxone and diprenorphine) with various phospholipid films (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and dioleoylphosphatidylglycerol). We were able to demonstrate the interaction of opiate drugs and opioid peptides (0.1 μM) with both zwitterionic and negatively charged phospholipids up to very high surface pressures. In the presence of a phospholipid monolayer, the surface activity of opioid peptides became much greater than that observed at the air-water interface. In the case of opiate drugs, the phenomenon was different and these drugs penetrate the monolayer films at a low pressure in comparison with opioid peptides, even at very high concentration. We have also evaluated the penetration of the antagonists naloxone and diprenorphine; the interaction of these was very low.

A study on human gastric lipase and complexes with monoclonal antibodies using the monomolecular film technique

Five monoclonal antibodies (MAbs) directed against human gastric lipase (HGL) were used as probes to characterize and locate the functional sites (catalytic and lipid binding domains) on the surface of the enzyme. Taking advantage of the low surface activity of IgG molecules, the capacity of the MAbs to inhibit the penetration of native and sulfhydryl (SH)-modified HGL into lipid monolayers was investigated using the monomolecular film technique. A schematic representation of the topography of the epitopes recognized by the MAbs in relation to the catalytic and lipid binding sites of the enzyme is proposed. The region containing the free SH group essential for lipase activity is identified.

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.

A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides

A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Role of a sulfhydryl group in gastric lipases. A binding study using the monomolecular-film technique.

Native human and rabbit gastric lipases (HGL and RGL, respectively) were inactivated after modification of one sulfhydryl group/enzyme molecule. HGL and RGL were covalently labeled using 5,5'-dithiobis(2-nitro-[14C]benzoic acid) and the interaction of 2-nitro-5-thio-[14C]benzoic-acid-labeled lipases ([14C]Nbs-lipases) with monomolecular lipid films was investigated. Our results show that [14C]Nbs-lipases bind to lipid films as efficiently as native HGL or RGL. The critical surface pressure pi c and the maximal surface pressure (delta pi max) of [14C]Nbs-lipases were enhanced in comparison with those of native RGL and HGL. These changes in behavior were probably due to an increase in hydrophobicity brought about, directly or indirectly, by the binding of the Nbs radical. This chemical modification thus blocks the hydrolysis site and reinforces the hydrophobic character of the gastric lipases.

Sterol-phospholipid interactions in model membranes Effect of polar group substitutions in the cholesterol side-chain at C 20 and C 22

The interactions of phospholipids with four different cholesterol derivatives substituted with one OH or one keto group at position C 20 or C 22 of the side-chain were studied. The derivatives were the 22, R-hydroxy; 22, S-hydroxy; 22-keto- and 20, S-hydroxycholesterol. Two aspects of the interactions were investigated: (1) the effect of the cholesterol derivatives on the gel → liquid crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) and of dielaidoylphosphatidylethanolamine (DEPE) monitored by differential scanning calorimetry and (2) The effect on the lamellar → hexagonal H II phase transition of DEPE monitored by DSC and by 31P-NMR to determine structural changes. The gel → liquid crystalline phase transition was affected by the cholesterol derivatives to a much larger extent in the case of DPPC than of DEPE. In both cases, there was a differential effect of the four derivatives, the 22, R-hydroxycholesterol being the less effective. In DPPC-sterol 1:1 systems, 22, R-hydroxycholesterol does not suppress the melting transition, the ΔH values becomes 7.1 kcal · mol −1 as compared to 8.2 kcal · mol −1 for the pure lipid. 22, S-OH cholesterol has a much stronger effect ( ΔH = 3.1 kcal · mol −1) and 22-ketocholesterol suppresses the transition completely. In DEPE mixtures of all these compounds, the melting transition of the phospholipid is still observable. The transition temperature was shifted to lower values (−13.5°C in the presence of 20, S-OH cholesterol). The ΔH of the transition was lowered by these compounds except in DEPE-22, R-OH cholesterol mixtures and the cooperativity of the transition (reflected by the width at half peak height) was reduced. The lamellar → hexagonal H II phase transition was also affected by the presence of these cholesterol derivatives. The transition temperature value was depressed with all these compounds. 20, S-OH cholesterol was the most effective followed by 22, R-OH cholesterol. The ΔH of the transition was not strongly affected. The molecular interfacial properties of these derivatives were studied by the monomolecular film technique. It is most likely that 22, R-OH cholesterol due to the hydroxyl groups at the 3β- and 22, R-positions orients with the sterol nucleus lying flat at the air/water interface, since the compression isotherm of either the pure sterol or the DOPC-sterol mixture (molar ration, 1:1) monomolecular film exhibits a transition at approx. 103 Å 2, corresponding to the area of revolution of the sterol nucleus. This remarkable property, due probably to the existence of a kink between the side-chain and the long axis of the steroid nucleus, might explain the smaller effect of this sterol on the melting transition of either PC or PE systems.

Surface-active properties of 1,2-distearolygalactosylglycerols

The surface-active properties of 1,2-distearoylmono- and digalactosylglycerols were examined by monomolecular film techniques and shadow-cast electron microscopy. The monogalactosyl lipid forms a typically condensed monolayer upon compression but at low surface densities a mosaic of condensed and liquid-expanded regions is formed, which is converted into a completely condensed film in a time-dependent process. The monolayer collapses by a fracture process at a surface pressure of about 50 mN · m −1 and multilayered, plate-like structures are observed. The digalactosyl lipid forms a completely liquid-expanded film at surface pressures less than about 15 mN · m −1 but becomes condensed on further compression. Monolayer collapse is seen at about 68 mN · m −1 but a biphasic relaxation process is detected in monolayers maintained at fixed surface areas when compressed to more than about 45 mN · m −1. Collapse of the monolayer at high surface pressure appears to take place by a nucleation and growth process and a mosaic of monolayer and multilayer structures can be seen in shadow-cast electron micrographs of films compressed to collapse surface pressures.

A possible orientation change of cardiotoxin molecule during its interaction with phospholipid monolayer

The monomolecular film technique was used in order to study the specific interactions of 4 cardiotoxins from Elapidae snake venom Naja mossambica mossambica with different phospholipids. The interaction, at pH 7.5, of cardiotoxin (10 -7M) with both neutral and negatively charged phospholipids occurs up to a very high critical surface pressure ( π = 45 dynes/cm with the latest). The apparent molecular area of cardiotoxin molecule, during its insertion into a negatively charged phospholipid film, presents only two characteristic values : 1400 Å 2 for π < 20 dynes/cm and 420 Å 2 for π > 30 dynes/cm , the transition occuring in a very narow range of surface pressure ( 25 ± 5 dynes/cm ). Thus, according to the surface pressure, the cardiotoxin may present two different orientations : “flat” or “edgewise” and the transition between both could account for lytic activity of cardiotoxin.

Penetration of phospholipid monolayers by cardiotoxins.

The monomolecular film technique was used to compare the specific interaction of four cardiotoxins from Naja mossambica mossambica with different phospholipids. We were able to demonstrate the interaction of cardiotoxins (10(-7) M) with both neutral and negatively charged phospholipids up to very high surface pressures (45 dyn/cm). In the presence of a phospholipid monolayer, the surface activity of cardiotoxins became much greater than that observed at the air-water interface. Neurotoxins of the same venom do not penetrate a phospholipid film, even at low surface pressure (15 dyn/cm). The apparent molecular area of cardiotoxin III during its insertion into a negatively charged phospholipid film was quantitatively defined. As a function of surface pressure of the membrane around 25 dyn/cm, cardiotoxins may exist in two different configurations, "flat" (1400 A2) or "edgewise" (420 A2). This result could account for the lytic activity of this type of toxin.

Ubiquinones have surface-active properties suited to transport electrons and protons across membranes.

Surface-active properties of ubiquinones and ubiquinols have been investigated by monomolecular-film techniques. Stable monolayers are formed at an air/water interface by the fully oxidized and reduced forms of the coenzyme; collapse pressures and hence stability of the films tend to increase with decreasing length of the isoprenoid side chain and films of the reduced coenzymes are more stable than those of their oxidized counterparts. Ubiquinone with a side chain of two isoprenoid units does not form stable monolayers at the air/water interface. Mixed monolayers of ubiquinol-10 or ubiquinone-10 with 1,2-dimyristoyl phosphatidylcholine, soya phosphatidylcholine and diphosphatidylglycerol do not exhibit ideal mixing characteristics. At surface pressures less than the collapse pressure of pure ubiquinone-10 monolayers (approx. 12mN.m(-1)) the isoprenoid chain is located substantially within the region occupied by the fatty acyl residues of the phospholipids. With increasing surface pressure the ubiquinones and their fully reduced equivalents are progressively squeezed out from between the phospholipid molecules until, at a pressure of about 35mN.m(-1), the film has surface properties consistent with that of the pure phospholipid monolayer. This suggests that the ubiquinone(ol) forms a separate phase overlying the phospholipid monolayer. The implications of this energetically poised situation, where the quinone(ol) is just able to penetrate the phospholipid film, are considered in terms of the function of ubiquinone(ol) as electron and proton carriers of energy-transducing membranes.

Studies of Lipase and Phospholipase A2 Acting on Lipid Monolayers

AbstractThe penetration process of various lipolytic enzymes into lipid monolayers was studied by the use of a surface barostat technique. The induction time, measured during pre‐steady state conditions, increased abruptly for a given value of the surface pressure. This appears to be due to a degree of lipid packing above which the enzyme no longer can penetrate the lipid film.Pancreatic lipase and phospholipase A2 have been shown by the monomolecular film technique to be progressively inactivated when adsorbed at the interface of their respective substrates. This inactivation is faster for lipase than for phospholipase. It is also enhanced by low film pressure and film transfer.The use of radioactive phospholipase and lipase samples offered the possibility to measure for the first time the amount of enzyme adsorbed at a monomolecular film. This adsorption was found to be relatively slow under the conditions of the assays.The existence of an optimum in the velocity versus surface pressure profile is the result of at least two counterbalancing factors. As the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the minimal specific activity of the enzyme increases.Using radioactively labelled lipase, we have shown for the first time in a bile salt free system that the critical surface pressure above which lipase can no longer penetrate a negatively charged monolayer is around 23 dynes/cm. This critical surface pressure is shifted up to 30 dynes/cm in the presence of colipase. The 23 to 30 dynes/cm range may be the critical region where colipase displays assistance during the penetration process of lipase under physiological conditions.From this study with monolayers, we can conclude that activity of lipolytic enzymes used as tools for probing biological membranes will be greatly influenced by the physicochemical nature of the membrane‐water interface.

Further studies of mode of action of lipolytic enzymes.

Pancreatic lipase and phospholipase A2 have been shown by the monomolecular film technique to be progressively inactivated when adsorbed at the interface of their respective substrates. This inactivation is faster for lipase than for phospholipase. It is also enhanced by low film pressures and film transfer. The use of radioactive phospholipase and lipase samples offered the possibility to measure the amount of enzyme adsorbed at a monomolecular film with a reasonable accuracy. This adsorption was found to be relatively slow under the conditions of the assays. The main conclusion drawn from these data is that the enzyme kinetics in presence of a substrate film, and probably also under bulk conditions, is controlled by an adsorption flux responsible for an initial lag period and an inactivation flux tending to decrease the reaction rate. The kinetics are linear only when both fluxes equilibrate.

Reduction of transpiration

The monomolecular film technique for reducing evaporation from water surfaces has been applied to the problem of transpiration from plants. Hybrid corn grown in soil enriched by various amounts of fatty alcohols such as hexadecanol has required up to 40 per cent less water during its growth than control plants. It is theorized that some of the transpiration from corn plants can safely be reduced by the blocking action of molecules of hexadecanol carried through plants and deposited at the stomate water-vapor interface. Although the blocking action could be taking place throughout the plants, testing with C14 hexadecanol has produced radioautographs showing activity not only in the roots and stalks but also throughout the leaves of treated corn plants. Comparative testing has been done in the greenhouse; however, one treated corn crop, grown naturally, yielded over 100 bushels per acre. Chemical tests on this corn showed no apparent effect of large doses of hexadecanol added to the roots of the plants.