Thermophilic proteins: stability and function in aqueous and organic solvents

The molecular stability of thermophilic and hyperthermophilic enzymes generally reflects the growth temperatures of the parent organisms. Extracellular enzymes from the hyperthermophilic Archaea typically show very high levels of thermal stability and a number of enzymes with Tm values of greater than 100 degrees C have been reported. The mechanisms responsible for high molecular stability are typically intrinsic characteristics of the protein, as shown by the comparative stabilities of many native and recombinant proteins. However, some extrinsic stabilisation mechanisms have been demonstrated. High levels of thermal stability are positively correlated with stability in the presence of other denaturing agents, including detergents and organic solvents. This correlation suggests a common denaturation pathway where molecular mobility/flexibility is the prime determinant of susceptibility to irreversible denaturation. In single phase organic-aqueous solvents, protein destabilisation occurs via solvent-induced alteration to the protein hydration shell. However, correlations between protein stability and solvent hydrophobicity are unreliable. In two-phase organic-aqueous systems, interfacial denaturation predominates and is a function of both interfacial tension and interfacial surface area. Intracellular enzymes are protected from interfacial denaturation but are potentially susceptible to direct organic solvent effects, possibly depending on the role of the cell wall and cell membrane in the partitioning of the organic solvent into the cell cytoplasm. Immobilisation of thermophilic enzymes provides a method for enhancing both the thermal and solvent stabilities of thermophilic and mesophilic enzymes. Multi-point covalent immobilisation to glyoxal-agarose enhances thermal stability and limits protein-protein inactivation mechanisms. Miscible organic solvents have a profound influence on the specificities of enzyme reactions. The presence of high concentrations of miscible organic solvents may induce gross changes in substrate specificity and/or more subtle alterations in chiral selectivity. Correlations between the variation in enantioselectivity and both solvent hydrophobicity and solvent dielectric constant have been demonstrated although some recent studies implicate the formation of specific solvent-enzyme complexes which directly affect reaction kinetics.     

Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells

The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum

Phospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Stv. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.     

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.     

Calculation of the interfacial properties of liquid steel – slag systems

The rates of many high temperature metallurgical reactions are strongly influenced by interfacial phenomena and to determine reaction rates it is necessary to have an accurate model of interfacial properties. In this article, the factors governing interfacial tension in slag-metal systems will be briefly discussed and, a model based on the combination of the Gibbs and Langmuir adsorption isotherms with Girifalco and Good's expression for the interfacial tension between immiscible liquids, will be shown to be consistent with current literature values and allow the calculation of both the interfacial tension and the contact angle between liquid slags and liquid iron in the CaO–CaF₂–SiO₂–Al₂O₃ system. The application of this approach to systems containing FeO in the slag and carbon in the metal are then discussed.     

Extraction kinetics of neodymium from chloride medium using HEH/EHP saponified with magnesium bicarbonate solution

Magnesium bicarbonate solution is considered as an environmentally friendly extractant saponification agent for the solvent extraction of rare earth elements due to its advantage of minimum water pollution.In order to reveal the extraction regularity, optimize production-process and guide the use of this new extraction system, the extraction of Nd(Ⅲ) in chloride medium with HEH/EHP saponified by magnesium bicarbonate solution was investigated with the self-designed constant interfacial area cell. Besides, the effects of stirring rate, temperature, specific interfacial area and concentration of Mg-HEH/EHP on the extraction rate of Nd(Ⅲ) were systematically investigated. Results show that, the rate of extraction is governed by both diffusion and chemical reaction, and the extraction reaction takes place at the interface. The apparent activation energy of the extraction reaction is 16.88 kJ/mol. The corresponding rate equation is deduced. The mechanisms and rate-determining step are speculated based on interfacial reaction models, which is consistent with the experimental results.     

Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.     

Hydrogen peroxide-induced phospholipase D activation in rat pheochromocytoma PC12 cells: possible involvement of Ca2+-dependent protein tyrosine kinase

The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.     

Self-evolving microstructured systems upon enzymatic catalysis

The consequences of cell microstructuration on enzyme functions is discussed in the framework of self-evolving microstructured systems. Molecular assemblies of amphiphiles or lipids are spontaneously formed by self-organisation. Among these different structures, reversed micelles, liquid crystalline mesophases and vesicles are hosts for enzymatic reaction studies. Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications; the catalytic behaviour of lipolytic enzymes, mainly phospholipase (PL) A2, is described in relation with structural aspects of biological membranes. The implication in cellular regulation events of PLC and PLD is discussed in relation with the role of their reaction products as second messengers in membrane fusion processes. The in vitro synthesis of dialkyl phosphatidylcholines, via the enzymatic 'salvage pathway' which leads to the formation of vesicles upon phospholipid formation, is considered in relation with autopoiesis. More recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OG/octanol/water phase diagram. Enzymatic formation of dipalmitoylphosphatidylcholine (DPPC) liposomes and non-ionic surfactant vesicles (NSV), starting from mixed micelles or open structures, finally sets an example of a biomimetic self-evolving system.     

Rate Processes in Solvent Extraction of Metal Ion

Abstract

The mechanism and kinetics of metal extraction with several extractants, including chelating agents, acidic organophosphorous extractant and neutral extractants are discussed in this paper.

Firstly the extraction rate of metal ion for the general extraction system is formulated by solving the diffusional equation with chemical reaction, in which the interfacial reaction is considered by the boundary conditions. The overall rate expressions of the complex formation with chelating agents are obtained both in aqueous phase and at the interface. The general formula of the extraction rate is simplified by considering the rate-determining steps.

Next, the extraction rate of metal ion with chelating agents and acidic organophosphorous extractants is discussed. For the chelating agents, the most important properly to determine the locus of complex formation is the distribution constant between the organic and aqueous phases. The contribution of the interfacial reaction to the extraction rate increases with the increase in the distribution constant of chelating agents. The extraction rate of many metal ions with acidic organophosphorous extractant are determined by the diffusion process of final complex in the organic phase.

Finally, the extraction rate of palladium ion with some neutral extractants is also discussed. The extraction rate is determined by the complex reaction in the aqueous phase and at the interface. The contribution of the interfacial reaction increases with the distribution constant of extractants.     

Implication of Ca(2+)-dependent protein tyrosine phosphorylation in carbachol-induced phospholipase D activation in rat pheochromocytoma PC12 cells

The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65-70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65-70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65-70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca(2+)-free buffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65-70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca(2+)-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Surface tension of binary metal—surface active solute systems under conditions relevant to welding metallurgy

Since the fluid flow, heat transfer, and the resulting weld properties are significantly affected by interfacial tension driven flow, the variation of interfacial tension in dilute binary solutions is studied as a function of both composition and temperature. Entropy and enthalpy of adsorption of surface active components such as oxygen, sulfur, selenium, and tellurium in Fe-O, Fe-S, Fe-Se, Cu-O, Cu-S, Cu-Se, Cu-Te, Ag-O, and Sn-Te systems were calculated from the analysis of the published data on interfacial tension of these systems. For these calculations, a formalism based on the combination of Gibbs and Langmuir adsorption isotherms was used. Interfacial tensions in Cr-O, Co-S, and Ni-S systems, where the data are scarce, were predicted by using certain approximations. The computed values were found to be in reasonable agreement with the data available in the literature. Temperature coefficients of interfacial tensions were calculated for several binary systems. It was demonstrated that in dilute solutions, the temperature coefficient of interfacial tension is strongly influenced by the heat of adsorption which, in turn, is influenced by the difference in electronegativity between the solute and solvent ions.     

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.     

Influence of chemical compositions of slag and graphite on the phenomena occurring in the graphite/slag interfacial region

Silica reduction reactions taking place in the slag/carbon interfacial region were investigated for synthetic/natural graphite in the temperature range 1500 °C to 1700 °C. Two silica-rich blast furnace slags, with low levels of iron oxide, were used in this study. Silica concentration in these slags, labeled as 1 and 2, was 30.80 pct and 36.80 pct with a respective basicity of 1.67 and 1.22. Reaction rate investigations were supplemented with wettability measurements on these systems with an aim to probe a possible interdependence between wetting characteristics and reaction rates of silica reduction. Wettability and slag/carbon reactions were studied in a horizontal tube resistance furnace in argon atmosphere, using the sessile drop approach. While the contact angles were measured by recording live images of the assembly with a charge-coupled device camera, the volumes of CO and CO₂ evolved were obtained from an analysis of off-gases with the help of a mass spectrometer. Reaction rates for silica reduction showed a wide variation for different systems. Synthetic graphite showed nonwetting behavior with both slags. Natural graphite, however, showed dynamic wetting with slag 2, resulting in low contact angles. This is attributed to the difference in the deposition of Si-based reaction products in the interfacial region, which in turn influences wettability. Temperature had a significant effect on both the wettability and silica reduction rate of the graphite/slag system. Activation energies for silica reduction in slags 1 and 2 with natural graphite were estimated to be 253 and 241 kJ/mol, respectively. Chemical composition of carbonaceous materials and slags were found to play a very important role in dictating overall reaction rates and wetting characteristics.     

Kinetics of interlayer formation on polycrystallineα-Al2O3/copper-titanium alloy interface

Copper-titanium alloys are known to wet an alumina surface. Traditionally, contact angles are used as measurable parameters to monitor the progress of wetting. Contact angle is a measure of the thermodynamic equilibrium at the interface. For reactive wetting systems, it is well known that the interfacial properties vary as a function of time. In the present study, kinetics of reaction were studied by monitoring the rate of interfacial phase formation. An immersion apparatus was built for this purpose. The extent of reaction was measured both by a simple surface compositional analysis and by interfacial reaction layer thickness measurements. The reaction layer exhibited a parabolic growth with an associated activation energy of 180 to 230 kJ/mole. A speculative growth mechanism is proposed based on the experimental observations and the information available in the literature.     

Poly(arylazophosphonate)s: A New Polymer Class with Arylazophosphonate Units in the Main Chain

Poly(arylazophosphonate)s are synthesized from bifunctional diazonium salts and bifunctional phosphoric diesters by interfacial polycondensation at the interface water/organic solvent. The azo unit is formed via N-P coupling during the polycondensation reaction in satisfying yield and selectivity. Fifteen new polymers were synthesized and characterized by common methods such as 1H, 13C, and 31P NMR, FTIR, UV/visible spectroscopy, and DSC and GPC measurements. According to first laser ablation experiments as well as film forming properties and thermal and photochemical behavior, poly(arylazophosphonate)s seem to be suitable materials for ablation with XeCl excimer lasers.     

Protein-nucleic acid recognition: simulation of base and "model" amino acids complexes in DMSO by the Monte Carlo method

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.     

Serodiagnosis of caseous lymphadenitis (pseudotuberculosis) in sheep

Random postmortal isolation of Corynebacterium pseudotuberculosis from the sheep used in a research project and the consequently determined serological positivity of these animals indicated the serological examination of the flock from which the sheep came. No symptoms of caseous lymphadenitis were ever observed in the flock in question and no data on the occurrence of this disease were available in the flock history. The sera of 228 head of sheep were used for examinations, while this number of sheep represented half the flock head. Agar-gel immunodiffusion (AID) and neutralization of the toxin C. pseudotuberculosis (NTX) were applied as serological assays. The antigen in both tests was an isolated form of toxin i.e. phospholipase D (PLD). To determine the effect of PLD, the intensity of its synergic hemolytic effect with the equi factor Rhodococcus equi was found out and it was expressed in activity units (AU). Undiluted serum with PLD containing 5,000 AU/ml was examined in AID. Positive precipitation was observed in 79 examined sera, out of which 71 were also positive in NTX. NTX demonstrated the inhibition of synergic hemolytic activity of PLD with the equi factor, which was used to sensibilize sheep erythrocytes in agar medium. PLD with the terminal content of 10 AU/ml was used, as well as twofold dilution series of sera. The first evaluated dilution series was 1:10. In this dilution series, the highest number of sera, 22, had the positive reaction. The highest positivity at a dilution series 1:5,120 was found out in one serum. A total of 71 sera had positive reactions in NTX and those sera were also positive in AID. The determined 34.7% positivity substantiated the importance of serodiagnosis for the intravital diagnosis of caseous lymphadenitis of sheep, particularly of its chronic form. AID and NTX are usable tests for serodiagnosis of this disease.