Illarion V. Turko

Protein tyrosine nitration in the mitochondria from diabetic mouse heart: Implications to dysfunctional mitochondria in diabetes

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.

Nitration of succinyl-CoA:3-oxoacid CoA-transferase in rats after endotoxin administration.

The tyrosine nitration of proteins has been observed in diverse inflammatory conditions and has been linked to the presence of reactive nitrogen species. From many in vitro experiments, it is apparent that tyrosine nitration may alter the function of proteins. A limited number of experiments under in vivo conditions also demonstrate that protein nitration is associated with altered cellular processes. To understand the association of protein nitration with the pathogenic mechanism of the disease, it is essential to identify specific protein targets of nitration with in vivo or intact tissue models. Using anti-nitrotyrosine antibodies, we demonstrated the accumulation of nitrotyrosine in a 52-kDa protein in rat kidney after lipopolysaccharide treatment. The 52-kDa protein was purified and identified with partial sequence as succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5). Western blot analysis revealed that the nitration of this mitochondrial enzyme increased in the kidneys and hearts of lipopolysaccharide-treated rats, whereas its catalytic activity decreased. These data suggest that tyrosine nitration may be a mechanism for the inhibition of SCOT activity in inflammatory conditions. SCOT is a key enzyme for ketone body utilization. Thus, tyrosine nitration of the enzyme with sepsis or inflammation may explain the altered metabolism of ketone bodies present in these disorders.

Direct antigen detection in Langmuir—Blodgett films of immunoglobulin G modified with coproporphyrin-I

Abstract A direct optical method based on the interaction of antigen with a thin film of immunoglobulin G (IgG) modified with a fluorescent label was developed for the fluorimetric determination of antigen—antibody binding. The analytical system uses a Langmuir—Blodgett film of coproporphyrin I-modified IgG which binds antigen and changes its fluorescent properties. The enhanced fluorescence is linear with respect to increasing concentration of antigen over a broad range. The main advantage of the proposed method is the possibility of developing a direct immunoassay based on evanescent excited fibre-optics.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen bindings, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

Antigen binding properties of Langmuir-Blodgett films of immunoglobulin G deposited onto the optical fiber core

Antigen binding properties and storage stability of immunoglobulin G (IgG) thin films deposited onto the quartz core of optical fibers are described.

Direct fluoroimmunoassay in Langmuir-Blodgett films of immunoglobulin G

4. I. V. Turko, I. 5, Yurkevich and V. L. Chashehin, Langniir—Blodgett Fi]iisof Iisainoglobilin G for limunosensors, Thin Solid Filis, in press, 1991.5. J. D. ArKIr&Ie, R. A. Vanwagenen, D. E. Gregonis, K. Neuby and J.-N. Lin,Fiber-.Ctic Biosensors Based on Evanescent-Excited Fluoro-lininoassay:Concept and Prcgress, IEEE Trans. a, Electra, Devices, vol. )-32, pp. 1175-1179,

Langmuir-Blodgett films of immunoglobulin G and direct immunochemical sensing

Langmuir-Blodgett technique is shown to be a suitable method for construction of the functionally-oriented layers of immunoglobulin G (IgG) which bind antigen better than in the case of chemical immobilization of IgG on the same substrate. It is assumed that the fluorescent detection of the conformational changes in the molecule of IgG or in the regular structures of IgG molecules after the interaction with antigen can be used for the development of the new type of immunosensors.