Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis.

An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM. The constitutive form of the enzyme was purified 312-fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine. The laccase was found to contain 3.9-4.1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1. Laccase was found to be a pH-stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3.5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.

[Selection of optimal conditions for synthesis of protein A-laccase conjugates and their use in different variants of immunoenzyme analysis]. / Podbor optimal'nykh uslovii sinteza kon''iugatov belok A-lakkaza i ikh ispol'zovanie v razlichnykh variantakh immunofermentnogo analiza.

A study to determine optimal molar ratios of Protein A to laccase for the synthesis of their conjugate by periodate method is presented. No loss of enzymatic or immunological activity of the conjugate developed was observed during 6 month. The conjugate could be effectively used in various techniques of enzyme immunoassay (competitive or sandwich techniques, dot immunoblotting) for immunoglobulin G. The detection limits of the assays for immunoglobulins were better than 1 ng/ml.

[Quantitative determination of cattle angiogenin]. / Kolichestvennoe opredelenie angiogenina byka.

Polyclonal antibodies to a milk antigen were obtained by a standard immunization procedure. The possibility was demonstrated to use the peroxidase-antibody conjugate in a competitive immunoenzymatic test. The minimal amount of the antigen determined by this method is 10 ng/ml.

[Immunoenzyme analysis based on laccase conjugates with fluorometric detection of the enzymatic reaction product]. / Immunofermentnyi analiz na osnove lakkaznykh kon''iugatov s fluorimetricheskoi detektsiei produkta fermentativnoi reaktsii.

The possibility of using homovanillic acid as a substrate of laccase (produced by the basidiomycete Coryolus hirsutus) has been demonstrated for the first time. The reaction was shown to result in the formation of a fluorescent product. Several kinetic parameters and optimal conditions were determined for this enzymatic reaction. The use of homovanillic acid as the substrate was found to increase the enzyme immunoassay sensitivity by an order of magnitude, compared to conventional substrates.

[Study of physiological functions of human ceruloplasmin. The effect of ceruloplasmin on immunocytes in a normal state and in pathology]. / Issledovanie fiziologicheskikh funktsii tseruloplazmina cheloveka. Vliianie tseruloplazmina na immunotsity v norme i pri patologii.

The immunomodulating effect of ceruloplasmin (CP) on the major components of the immunocompetent system of the organism--the natural resistance system and the specific immune response--has been established. CP can exert various influences on the level of expression of specific markers of T- and B-lymphocytes (as determined by various modifications of the rosette-forming test), on the phagocytic activity of neutrophils and monocytes as well as on the activity of "respiratory burst" enzymes. CP modulation was found to depend predominantly on the initial level of the immunological parameters to be determined, i. e., on the extent of immune inflammation in human patients. Thus, it was found that CP not only plays the roles of an antioxidant and a copper-transporting protein, but is also capable to interact with immunocytes, altering their biological activities. The observed immunotropicity of CP, its ability to directly interact with immunocytes and to model the immune function at the cell level provides evidence for the existence of a universal molecular language of information exchange between the macroorganism cells of various nature and origin.

[The effect of conditions of synthesis of immunolaccase conjugates on characteristics and composition of the compounds obtained]. / Vliianie uslovii sinteza immunolakkaznykh kon''iugatov na kharakteristiki i sostav poluchaemykh soedinenii.

Optimal conditions for preparing laccase conjugates by the periodate method have been selected. The effect of the initial molar ratio of IgG to laccase and pH of the medium on the composition of laccase conjugates was studied by the HPLC method. The maximum yield of the conjugates was observed, when laccase was oxidized with 0.12 M sodium periodate the pH of the medium was 8.5, and the initial molar ratio of IgG to laccase was 2:1. The conjugates can be stored for one year without any loss in immunological activity.

Modulatory effects of ceruloplasmin on lymphocytes, neutrophils and monocytes of patients with altered immune status.

We investigated the effect of plasma ceruloplasmin (Cp) on the different types of lymphocyte rosetting, and phagocytosis of polystyrene particles and culture Candida albicans by peripheral blood neutrophils and monocytes. Lymphocytes, neutrophils, and monocytes were isolated from the blood of patients with elevated immuno-status (n = 9), healthy donors (n = 21), and patients with reduced immuno-status (n = 21). The ability of Cp to decrease the number of lymphocytes forming E- and EAC-rosettes and rosettes with auto-erythrocytes was shown for both patients and healthy donors. The maximal decrease of the number of E-rosettes (by 35%) and EAC-rosettes (by 57%) was shown for lymphocytes of the patients with elevated immuno-status. Cp had an effect on rosetting only when lymphocytes were preincubated with it, suggesting that Cp binding to lymphocytes was responsible for these effects. The decrease in all types of rosetting caused by Cp was dose-related, with a maximum effect at physiological concentration of Cp (300 micrograms/ml). We demonstrated an enhancing effect of Cp on phagocytosis of Candida albicans and polystyrene particles by neutrophils (with a maximum enhancement by 180% for neutrophils of the patients with decreased immuno-status) and monocytes (with a maximum of 89% for monocytes of healthy donors). Cp enhances phagocytosis of neutrophils and monocytes by binding these cells, not by opsonizing ingested particles as a conventional opsonin (ie. lipopolysaccharide from E.coli). The stimulating effect of Cp on phagocytosis was three times higher than that of LPS from E.coli.

[Laccase from Coriolus hirsutus--a new marker enzyme for immunoenzyme analysis]. / Lakkaza Coriolus hirsutus--novyi ferment-marker dlia immunofermentnogo analiza.

A new immunochemical reagent is proposed which contains laccase, isolated from the culture liquid of the basidial fungus Coriolus hirsutus, as a marker enzyme. The feasibility of immunolaccase conjugates for different variants of immunoassay, i.e. "sandwich", competitive and indirect, is demonstrated. The comparison of immunolaccase and immunoperoxidase conjugates showed that the absolute sensitivity of laccase-antibody conjugates was 3 times higher than that of antibody-peroxidase conjugates (7.7 x 10(-11) M and 2.3 x 10(-10) M, respectively). The assay based on antibody-laccase conjugates is simpler than that employing antibody-peroxidase conjugates, since in the former case air oxygen in used as the second substrate of the enzymatic reaction.

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode. The formation of the complex between laccase-labelled antibody and antigen on the electrode surface resulted in a considerable (more than 300 mV) shift of the electrode potential. The rate of the increase of the electrode potential was inversely proportional to the concentration of the free antigen in the sample. The non-specific adsorption of conjugate and other proteins on the electrode could be eliminated by using a polyethylenimine-based polymer on the electrode surface. Insulin was used as a model analyte. The sensitivity limit for this antigen was approximately 3 micrograms ml-1.

[Interconnection between the structure and protective action of normal and pathological ceruloplasmin preparations in copper-induced erythrocyte lysis]. / Vzaimosviaz' mezhdu stroeniem i zashchitnym deistviem preparatov normal'nogo i patologicheskogo tseruloplazmina pri med'indutsirovannom lizise éritrotsitov.

It was found that the differences in the protective effects of ceruloplasmin (CP) isolated from the blood of healthy donors and of the ceruloplasmin-like protein (pat-CP) isolated from the blood of patients with hepatovertebral dystrophy (HCD) during Ca(2+)-induced lysis of erythrocytes (RBC) result from significant changes in the carbohydrate fragment of pat-CP, the bulk of which (65%) is devoid of mannose and acetylglucosamine residues. According to the data from lentil-lectin Sepharose chromatography, only 4% of pat-CP molecules contain the [formula; see text] fragment necessary for the binding to ER receptors. The curves reflecting the Cu2+ accumulation in healthy donor ER and in pat-CP during the Cu(2+)-induced lysis were found to differ significantly. The ability of pat-CP to prevent the accumulation of Cu2+ in ER and pat-ER was markedly decreased compared with CP. Besides, CP prevented the diminution of reduced glutathione (GSH) in ER in a greater degree than pat-CP, whereas pat-ER, in contrast with CP, had no effect on the GSH concentration in pat-ER. It is suggested that the reactions occurring in the cell during Cu(2+)-induced lysis of ER and pat-ER are different.

Immunopotentiometric electrodes based on bioelectrocatalysis in the absence of mediators.

A new method of immunoelectrochemical analysis employing laccase as the enzyme label is described. The ability of the enzyme to catalyze electroreduction of oxygen via a direct mechanism allows the detection of the biospecific interaction of a laccase-labeled receptor, or antibody, with a ligand-modified electrode. Formation of a complex between the laccase-labeled antibody and the antigen on the electrode surface resulted in a considerable (greater than 300 mV) change in the electrode potential. Analysis was performed in a competitive scheme, and a single measurement could be made within 20 min in the absence of an electrochemically active mediator. The reaction substrates were atmospheric oxygen and electrons that were transferred directly from the electrode to the active site of the enzyme label. The use of a composite carbon material containing a polyethyleneimine-based polymer eliminated nonspecific interactions between the reaction components and the electrode surface. Insulin and mouse immunoglobulin were used as a model analytes.

Interrelation between structure and protective action of normal and pathological ceruloplasmins during copper-induced lysis of red blood cells.

The origin of the difference between the protective action of ceruloplasmin (CP) from healthy donors blood and of ceruloplasmin-like protein (p-CP) from blood of patients with Wilson disease which they exert during copper-induced lysis of red blood cells (RBC) was elucidated. The difference is due to a significant change in the carbohydrate moiety of p-CP the major proportion of which (65%) does not contain mannose and acetylglucosamine residues. The data of chromatography on lentil lectin reveal that only 4% of p-CP molecules contain the fragment [table: see text] required for binding to RBC receptors. It was shown that the time-courses of copper accumulation in RBC of normal donors and in RBC of patients with Wilson disease (p-RBC) during copper-induced lysis differ markedly from each other. The p-CP is able to prevent copper accumulation in RBC and p-RBC to a significantly less degree than CP. It was also established that CP prevents the decrease of reduced glutathione (GSH) level in RBC to a greater extent than p-CP. In contrast to CP, the p-CP exerts no effect on the decrease in GSH concentration in p-RBC. These results may indicate that no interaction between Cu2+ and reduced glutathione takes place in p-RBC, in contrast to the situation occurring in normal RBC.

[Protective effect of ceruloplasmin during human erythrocyte lysis induced by Fe2+ ions]. / Zashchitnoe deistvie tseruloplazmina pri lizise éritrotsitov cheloveka, indutsirovannom ionami zheleza.

In order to elucidate the nature of linkage between the oxidase activity and protective effect of ceruloplasmin during the Fe2(+)-induced lysis of erythrocytes, the both factors were identified in ceruloplasmin samples prepared from blood sera of healthy donors and patients with hepatocerebral dystrophy (HCD). It was found that the oxidase activity of healthy donor ceruloplasmin markedly exceeds that of HCD patients, whereas the protective effect of the HCD protein, contrariwise, markedly exceeds that of normal ceruloplasmin. The data obtained suggest that the protective effect of ceruloplasmin during Fe2(+)-induced erythrocyte lysis is not correlated with its oxidase (ferroxidase, in particular) activity.

The protective effect of normal and pathological (Wilson disease) ceruloplasmins on human erythrocytes.

The binding of the ceruloplasmin (CP) from the healthy donor blood and of ceruloplasmin-like protein (p-CP) isolated from the Wilson patients' blood with erythrocytes (RBC) of healthy donors and with RBC of Wilson's patients (p-RBC) was investigated. It was shown, that the number of CP binding sites both on the RBC and p-RBC was significantly lower than that for p-CP, but Kd value for p-CP binding with both types of erythrocytes was approximately ten times higher than Kd value for CP. The protective action of CP on copper stimulated hemolysis is significantly higher than that of p-CP. The protective action of CP on ferrous ion stimulated hemolysis does not correlate with its ferroxidase activity. Contrariwise, the protective effect of p-CP which has no ferroxidase activity is more powerful than that of CP.

Protective action of blood ceruloplasmin obtained from normal individuals on red blood cells compared with that from patients with Wilson's disease.

A ceruloplasmin-like protein (pathological CP, p-CP) was isolated from the serum of patients with Wilson's disease whose antigenic determinant was identical to that of ceruloplasmin (CP) obtained from the blood of normal individuals. Because the protective action of CP involves its interaction with receptors on red blood cells (RBC), the binding of CP and p-CP to the RBC from normal individuals and to those from patients with Wilson's disease (p-RBC) was investigated. The number of binding sites for CP on both types of RBC was significantly lower than that for p-CP. The dissociation constants for CP-RBC and CP-p-RBC complexes were very similar (1.15 and 1.37 nM, respectively), as were those for the complexes of p-CP with RBC and p-RBC (11.1 and 11.8 nM, respectively), but the binding constants for p-CP with RBC of both types were ten times higher than the CP binding constants. The ability of CP to prevent Cu(2+)-induced lysis of RBC was significantly higher than that of p-CP. The protective action of CP and p-CP during RBC lysis in two Fe(2+)-containing systems did not correlate with their oxidative and ferroxidase activities. On the contrary, the protective effect of p-CP, which had significantly lower oxidase activity and no ferroxidase activity, was greater than that of CP.

[Protective effect rendered by human ceruloplasmin on erythrocytes in hepatocerebral dystrophy]. / Zashchitnoe deistvie, okazyvaemoe tseruloplazminom chekoveka na éritrotsity pri gepatotserebral'noi distrofii.

In order to elucidate the protective effect of human ceruloplasmin (CP) on erythrocytes in patients with hepatocerebral dystrophy (HCD), the parameters reflecting the interaction of CP from the blood of healthy donors (n-CP) and of HCD patients (h-CP) with erythrocytes from healthy donors (n-ER) and from HCD patients (h-ER) were estimated. The protective effects of n-CP and h-CP on n-ER and h-ER during the Cu2+-induced lysis were compared. It was shown that the ability of h-CP to prevent the human ER breakdown upon Cu2+-induced lysis is much lower (approximately 3-fold) than that of n-CP. The differences in the protective effect of n-CP and h-CP are manifested in a greater degree during the n-ER lysis than during the h-ER lysis.