Thermal stability of lactate dehydrogenase in the complex with the anion polyelectrolyte poly(styrene sulfonate)

The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

A special role of phosphate in the stability of lactate dehydrogenase against destruction by a polyelectrolyte

It has been shown by X-ray structure analysis that proteins have specific anion-binding sites for sulfate, citrate, and phosphate ions; however, the functional role of these anions is not always clear. Thus, it is unknown which of two phosphate anions, mono- or divalent, determines the stability of cellular proteins under stress conditions. In the present work, the influence of phosphate, sulfate, and chloride on the stability of lactate dehydrogenase (LDH) in the presence of poly(styrenesulfonate) (PSS) has been investigated by the methods of steady-state kinetics and intrinsic protein fluorescence. The study is based on the analysis of differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of the concentrations of mono- and bivalent phosphate forms differs, whereas sulfate remains in the bivalent form. It was shown that the differences between the influence of phosphate and sulfate ions at pH 7.0 were greater; divalent phosphate ions much more effectively stabilized LDH against destruction by a polyelectrolyte compared with sulfate and monovalent phosphate. It was concluded that, of two anion-binding sites of the LDH molecule, the intersubunit center plays the most important role in its stabilization against destruction by polyelectrolyte, and, of two forms of phosphate anions, its bivalent form HPO ₄ ^?2 plays the stabilizing role.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

The role of poly(ethyleneimine) in stabilization against metal-catalyzed oxidation of proteins: a case study with lactate dehydrogenase

The protection provided by poly(ethyleneimine) (PEI) to muscle lactate dehydrogenase (LDH) in metal-catalyzed oxidation (MCO) systems (CuSO(4) or FeCl(2) combined with H(2)O(2)) was studied, and comparisons were made with the chelators EDTA and desferal, respectively. The analytical chelating capacity of PEI was estimated to be around 1 mol Cu(2+)/10 mol ethyleneimine for all molecular weights of the polymer. The effect of [PEI monomer]/[metal ion] molar ratio on the oxidatively induced aggregation of LDH exhibited a similar trend as that of the other chelators; aggregation was enhanced at lower ratios and subsequently decreased until it was undetectable with increasing ratio. In contrast, the LDH activity showed a monotonic increase with increasing concentrations of the chelator. Total protection to the enzyme by PEI was provided at concentrations lower than that needed for full chelation of the copper ions, i.e. at [PEI monomer]/[Cu(2+)] ratio above 9 in case of PEI 2000, and above 7 for PEI 25000 and 2.6 x 10(6), respectively. The polymer also provided protection against oxidation in an iron-based MCO system. Hydroxyl radical formation during the MCO reaction was inhibited in the presence of PEI. The polymer of higher molecular weights also exhibited a stronger free radical scavenging effect.     

Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels]

The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.     

Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase.

1. The pH-dependencies of the binding of NADH and reduced nicotinamide--benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3--6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.     

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.     

Direct measurement of proton binding to the active ternary complex of pig heart lactate dehydrogenase (Short Communication)

With the use of a glass electrode it is shown that one proton is taken up from the solvent when o-nitrophenylpyruvate forms a ternary complex with lactate dehydrogenase and NADH at pH8 and before the complex has rearranged to give products.     

Stable stealth function for hollow polyelectrolyte microcapsules through a poly(ethylene glycol) grafted polyelectrolyte adlayer

Prospective biomedical applications of hollow polyelectrolyte microcapsules, for example, as drug delivery systems, require surface modifications that help to escape clearance by the mononuclear phagocytic system (MPS). Layer-by-layer assembled microcapsules that were alternatingly composed of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) were coated with adlayers of poly(ethylene glycol) (PEG)-grafted poly-L-lysine (PLL-g-PEG) and poly-L-glutamic acid (PGA-g-PEG). Their effects on MPS recognition were studied in primary cell cultures of human monocyte derived dendritic cells and macrophages. PGA-g-PEG coatings had no significant effect on cellular recognition, which may be explained by insufficient PEG density of the adlayer. Contrary, PLL-g-PEG effectively blocked phagocytosis of coated microcapsules. In addition, PLL-g-PEG coatings showed efficient adlayer stability for at least 3 weeks, and PAH/PSS microcapsules did not impair phagocyte viability. Our results demonstrate that layer-by-layer assembled polyelectrolyte microcapsules coated with a PEG-grafted polyelectrolyte, PLL-g-PEG, represent a promising platform for a drug delivery system that escapes fast clearance by the MPS.     

Complexation of trypsin and alcohol dehydrogenase with poly(diallyldimethylammonium chloride)

Complexation of alcohol dehydrogenase (ADH) and trypsin with poly(diallyldimethyl-ammonium chloride) (PDADMAC) in dilute electrolyte solution was studied by turbidimetric titration, quasi-elastic light scattering (QELS), and electrophoretic light scattering (ELS). Both QELS and turbidimetric titration show that PDADMAC forms complexes with ADH and trypsin in 0.01M NaCl solution at pH ≥ 6.8 and pH ≥ 9.2, respectively. These complexes take the form of stable coacervates in 0.01M, pH 11.0, phosphate buffer solution. QELS shows sizes of 400 and 315 nm for the coacervates of ADH-PDMDAAC and trypsin-PDMDAAC, respectively, while ELS reveals that these coacervates carry a net positive charge. Activity measurements show that both ADH and trypsin are enzymatically active in their coacervated states. Complexation of trypsin and PDADMAC was also studied by fluorescence in 0.01M, pH 11.0, phosphate buffer, and the protein emission was found to be quenched by complexation. The fluorescence quenching data show that trypsin retains its three-dimensional structure in the complex. These and other results are consistent with the quenching of the two tryptophans on the protein surface, but not the interior ones.© 1997 John Wiley & Sons, Inc.     

Colonial breeding in Mute swans (Cygnus olor) associated with an allozyme of lactate dehydrogenase

The Mute swan ( Cygnus olor Gmelin (Anatidae)) is a common water bird of lowland freshwaters and coastal shallows. Its typical breeding system involves lifelong monogamous pairs which vigorously defend large breeding territories, sometimes killing intruding swans that are unable to escape. However, in some unusual circumstances (superabundant food coupled with limited nesting sites) Mute swans may nest colonially. At the only two colonies in southern England a rare allele for lactate dehydrogenase (LDH) was found to be unusually common and colony swans carrying this allele were shown to breed more successfully. This isolated finding could, however, have originated either from a chance 'founder' effect or from human management of the main colony. We now show that this allele is also significantly commoner at two recently formed colonies in Denmark, implying that the association between the allele and colonial breeding may be widespread and longstanding.