A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.     

Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes.

A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered.     

Simple dissolution-reaction model for enzymatic conversion of suspension of solid substrate

Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the alpha-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (k(L)), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, k(L) may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.     

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.     

The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region.

The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.     

Subunit interactions in the methionyl-tRNA synthetase of Bacillus stearothermophilus.

The methionyl-tRNA synthetase from Bacillus stearothermophilus is shown to be a dimer of 2 x 82,000 with identical subunits. It exhibits negative cooperativity in substrate binding and "virtual" halt-of-the-sites reactivity. The enzyme binds only 1 mol of methionine in the absence of other ligands, but several methods show that 2 mol of methionyl adenylate are bound per enzyme dimer. However, one of these adenylates is formed 480 times faster than the other (k1 = 29 sec-1 and k2 = 0.06 sec-1). While the rapid phase of the reaction follows normal saturation kinetics with respect to substrate concentration, the rate of the slow phase is independent of substrate concentrations down to 1 muM. It is suggested that the very slow rate of formation of the second adenylate reflects a rate limiting conformational change which precedes a more rapid chemical step on the second subunit.     

A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.     

Effect of a diazafluoranthen derivative on phospholipases A study at the air-water interface

AC-3579 (2-N-methylpiperazinomethyl-1,3-diazafluoranthen 1-oxide) produces in rat hepatocytes a hypertrophy of the endoplasmic reticulum.Two possibilities that can explain this phenomenon are (1) that AC-3579 inactivates the phospholipases, and (2) that an AC-3579-lipid interaction hinders the enzymic activity.To demonstrate these hypotheses, a physicochemical model of biological membrane, the lipid-water interface, has been used. Dipalmitoyl dl-α-phosphatidylcholine was spread at the air-water interface, the enzymes (phospholipase A or phospholipase C) dissolved in the aqueous phase.The enzymic reaction was first studied with and without AC-3579 dissolved in the aqueous phase; no enzymic inactivation was observed. However in AC-3579- lipid complex completely inhibited the enzymic reaction in the case of phospholipase A.An explanation is given in terms of steric hindrance to the enzyme-substrate complex formation.     

Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

Effect of a diazafluoranthen derivative on phospholipases. A study at the air-water interface.

AC-3579 (2-N-methylpiperazinomethyl-1,3-diazafluoranthen 1-oxide) produces in rat hepatocytes a hypertrophy of the endoplasmic reticulum. Two possibilities that can explain this phenomenon are (1) that AC-3579 inactivates the phospholipases, and (2) that an AC-3579-lipid interaction hinders the enzymic activity. To demonstrate these hypotheses, a physicochemical model of biological membrane, the lipid-water interface, has been used. Dipalmitoyl DL-alpha-phosphatidyl-choline was spread at the air-water interface, the enzymes (phospholipase A or phospholipase C) dissolved in the aqueous phase. The enzymic reaction was first studied with and without AC-3579 dissolved in the aqueous phase; no enzymic inactivation was observed. However an AC-3579-lipid complex completely inhibited the enzymic reaction in the case of phospholipase A. An explanation is given in terms of steric hindrance to the enzyme-substrate.     

The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k(red) (the limiting rate of enzyme reduction by substrate at high [S]) decreased approximately 20-fold in an R310K variant and 2 x 10(4)-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed approximately 4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k(red) greater than 10(3)), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k(red) less than 50-fold). The effect was such that the 4000-fold range in k(red) seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.     

Rapid-scan stopped-flow studies of the flavoenzyme mercuric reductase during catalytic turnover

Time-resolved absorption spectra of the FAD-containing enzyme mercuric reductase were recorded during the catalytic reaction at 25 degrees C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k = 43 s-1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two-electron-reduced enzyme, EH2-NADPH. This spectrum persisted during the quasisteady-state phase of the reaction suggesting that EH2-NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2-NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2-NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two-electron-reduced enzyme, EH2-NADP+. These results suggest that EH2-HADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle.     

Cholesterol oxidase senses subtle changes in lipid bilayer structure

We investigated the dependence of cholesterol oxidase catalytic activity and membrane affinity on lipid structure in model membrane bilayers. The binding affinities of cholesterol oxidase to 100-nm unilamellar vesicles composed of mixtures of DOPC or DPPC and cholesterol are not sensitive to cholesterol mole fraction if the phase of the membrane is in a fluid state. When the membrane is in a solid-ordered state, the binding affinity of cholesterol oxidase increases approximately 10-fold. The second-order rate constants (kcat*/Km*) for different lipid mixtures show a 2-fold substrate specificity for cholesterol in the l(d) phase of high cholesterol chemical activity over cholesterol in the l(o) phase. Moreover, the enzyme is 2-fold more specific for cholesterol in the l(o) phase than in the s(o) phase. Likewise, there is 2-fold substrate specificity for the high cholesterol chemical activity l(d) phase over the low chemical activity l(d) phase. The specificities for the l(d) phase of low cholesterol chemical activity and the l(o) phase are the same. These data indicate that the more ordered the lipid cholesterol structure in the bilayer, the lower the catalytic rate. However, under all of the conditions investigated, the enzyme is never saturated with substrate. The enzymatic activity directly reflects the facility with which cholesterol can move out of the membrane, whether changes in cholesterol transfer facility are due to phase changes or more localized changes in packing. We conclude that the activity of cholesterol oxidase is directly and sensitively dependent on the physical properties of the membrane in which its substrate is bound.     

Transgalactosylation in a water-solvent biphasic reaction system with beta-galactosidase displayed on the surfaces of Bacillus subtilis spores

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that beta-galactosidase (beta-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 x 10(3) U/g (dry weight) of spores. The beta-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed beta-Gal, octyl-beta-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.     

Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.     

The catalytic power of enzymes: conformational selection or transition state stabilization?

The mechanism by which enzymes produce enormous rate enhancements in the reactions they catalyze remains unknown. Two viewpoints, selection of ground state conformations and stabilization of the transition state, are present in the literature in apparent opposition. To provide more insight into current discussion about enzyme efficiency, a two-state model of enzyme catalysis was developed. The model was designed to include both the pre-chemical (ground state conformations) and the chemical (transition state) components of the process for the substrate both in water and in the enzyme. Although the model is of general applicability, the chorismate to prephenate reaction catalyzed by chorismate mutase was chosen for illustrative purposes. The resulting kinetic equations show that the catalytic power of enzymes, quantified as the k(cat)/k(uncat) ratio, is the product of two terms: one including the equilibrium constants for the substrate conformational states and the other including the rate constants for the uncatalyzed and catalyzed chemical reactions. The model shows that these components are not mutually exclusive and can be simultaneously present in an enzymic system, being their relative contribution a property of the enzyme. The developed mathematical expressions reveal that the conformational and reaction components of the process perform differently for the translation of molecular efficiency (changes in energy levels) into observed enzymic efficiency (changes in k(cat)), being, in general, more productive the component involving the transition state.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.     

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.     

Lipid-water interface mediates reversible ionophore conformational change

A new procedure of conformational analysis was used to demonstrate that the ionophore conformation is mediated by its membrane environment. In the hydrophobic lipid matrix, the ionomycin-Ca++ complex adopts a conformation well suited for translocation across the interior of the membrane whereas at the lipid-water interface, the Ca++ ion is immersed into the aqueous phase in a position favorable to its complexation or decomplexation. The translocation of Ca++ across the lipid bilayer supposes a reversible transformation of the two conformers. The conformational analysis shows how the dielectric constant discontinuity existing at the lipid-water interface mediates the reversible transformation of one structure into the other.