Activity of purified pyruvate dehydrogenase and 2 - oksoglutarate dehydrogenase multienzyme complexes was studied and a quantity of free SH-groups in them being influenced by chlorides of cadmium, copper and lead was determined. The conducted research proved a considerable activity impairment of dehydrogenases of oxoacids and of the free SH – groups level

Budnyak, Oleksander K.

Petrov, Sergiy A.

Semenova, Olga O.

English

European Scientific Journal, ESJ

European Scientific Journal November 2014 /SPECIAL/ edition vol.2 ISSN: 1857 – 7881 (Print) e - ISSN 1857- 7431 200 THE MECHANISMS INHIBITING PYRUVATE- AND OXOGLUTARATE  DEHYDROGENASE COMPLEXES IN MUSSELS    Olga O. Semenova, PhD Student Ukrainian Scientific Research Institute of Transport Medicine, Ukraine Oleksander K. Budnyak, Assoc. Prof., PhD Sergiy A. Petrov, Full Prof. Odessa I.I. Mechnikov State University, Ukraine   Abstract  Activity of purified pyruvate dehydrogenase and 2 - oksoglutarate dehydrogenase multienzyme complexes was studied and a quantity of free SH-groups in them being influenced by chlorides of cadmium, copper and lead was determined. The conducted research proved a considerable activity impairment of dehydrogenases of oxoacids and of the free SH – groups level.  Keywords: Heavy metals, activity of multienzyme complexes, SH-groups level  Introduction The world scientific literature contains a considerable bulk of data describing a study of the impact which heavy metals produce on marine aquatic organisms, particularly on mussels, and accumulation of certain substances in their body tissues, survival of marine aquatic organisms, impact on their physiological functions and activity of various enzymes, etc. (Bren’ 1999; Soldatov et al., 2005; Gostyukhina, 2005).  However, the influence of heavy metal compounds upon the activity of the purified dehydrogenase of the Krebs cycle was studied in the individual works only. Therefore, the purpose of our work was to study the activity of purified pyruvate dehydrogenase (PDC) and 2 - oksoglutarate dehydrogenase (OGDC) multi-enzyme complexes isolated from the hepatopancreas of mussels, and the level of free SH - groups in them being influenced by chlorides of such heavy metals as cadmium, copper and lead (Cu, Cd, Pb).  Materials and methods We selected the Black Sea mussels Mytilus galloprovincialis Lam. Black morphs of 4-5 cm size. The adaptation period equalled 5 days and afterwards the mussels were used for experiments. Effects of various concentrations (0.1, 1.0 and 10.0 mg/l) of Cu, Cd and Pb chlorides  upon the activity of purified mussel hepatopancreas according to (Roche & Cate, 1977) dehydrogenase oxoacids of Krebs cycle (the activity was determined by Potassium ferricyanide method (Kiessling & Lunquist, 1962), and the concentration of SH - groups - by the method using the Ellman’s reagent (Orekhovych, 1977). The obtained data was statistically processed according to S. Glantz (Glantz, 1998).   Results The results of the research are presented in Table 1.  As follows from Table 1, the activity of purified  PDC and OGDC reduced pro rata when exposed to the increasing concentrations of CdCl2 and PbCl2; as regards the action of European Scientific Journal November 2014 /SPECIAL/ edition vol.2 ISSN: 1857 – 7881 (Print) e - ISSN 1857- 7431 201 CuCl2 in the incubated environment, the activity of both enzyme complexes was strongly inhibited at all concentrations, especially of the OGDC at 1 mg/l concentration.  Having obtained the data that confirmed a considerable inhibition of the PDC and OGDC activity by heavy metal chlorides, we decided to clarify a possible mechanism of this phenomenon. It is well known that many heavy metals found in tissues of humans and mammals can inhibit activity of a diversity of enzymes due to blocking their SH-groups that participate in the catalytic process either directly or indirectly.  Table 1. Activity of purified pyruvate dehydrogenase complex (PDC) and 2-oxoglutarate dehydrogenase (OGDC) multienzyme complexes and concentration of SH-groups in hepatopancreas of the Black Sea mussels Mytilus galloprovincialis Lam. being  influenced by Cu, Cd and Pb chlorides in 0.1, 1.0 and 10 0 mg / l concentrations   PDC OGDC   Activity, nM K3[Fe(CN)6]/mg protein/min SH-groups, nM/mg protein Activity, nM K3[Fe(CN)6]/mg protein/min SH-groups, nM/mg protein Control - 263.19±5.72 67.76±3.54 266.46±1.39 75.46±3.77 Cd mg/ml 0.1 210.55±8.55* 47.37±1.09* 243.26±10.83 52.69±0.40*  1 124.73±8.86* 21.05±0.96* 168.59±6.28* 33.50±0.40*  10 88.11±7.24* 13.82±0.40* 101.83±7.66* 14.31±0.61* Сu mg/ml 0,1 35.47±6.35* 8.68±0.32* 202.53±7.80* 44.50±0.64*  1 26.32±7.60* 7.10±0.34* 16.97±6.83* 1.76±0.24*  10 32.04±6.85* 7.90±0.32* 43.00±3.72* 11.38±0.52* Pb mg/ml 0,1 127.02±7.53* 31.25±0.52* 168.59±7.80* 31.44±0.84*  1 107.57±5.91* 24.67±0.74* 118.80±10.83* 26.67±0.40*  10 34.33±9.34* 8.88±0.40* 61.10±6.28* 17.24±0.40* Note: * significant difference from control group equals р≤0.05.  Therefore it was necessary to investigate whether a similar phenomenon takes place in case of enzymes of the mussel tissue. To this end, we determined the number of SH-groups in the purified enzymes of the control group and in the presence of the cadmium, copper and lead salts (Table 1). According to the obtained data, the salts of all investigated heavy metals reduced the content of SH-groups in the enzymes in a dose-dependent manner, following the almost similar regularities that govern the changes in their activity.   Conclusion The conducted study has proved that at the level of the purified enzymes of PDC and OGDC copper exhibited the highest inhibitory activity as compared to other studied metals – cadmium and lead. Inhibition of the PDC and OGDC activity occurs due to blocking SH-groups of enzymes.  References: Bren’ N.V. (1999) Use of the invertebrates for monitoring pollution of aquatic ecosystems with heavy metals (Review). Gidrobiol. journal. 35: 75 – 87. Glantz S. Medical and biological statistics. Transl. from Eng. – Moscow, Praktika, 1999. - 460 p.  Gostyukhina O.L. (2005) Condition of the enzyme system of antioxidant protection in Black Sea mussels Mytilus galloprovincialis Lam. under conditions of the natural oxidative stress: brown morpha. Scientificproceedings of Ternopol National Ped. University, series "Biology". Special issue: Hydroecology 27: 52 – 54.  European Scientific Journal November 2014 /SPECIAL/ edition vol.2 ISSN: 1857 – 7881 (Print) e - ISSN 1857- 7431 202 Soldatov А. А., Aleksandrova О. L., Golovina [et al.] (2003) Enzyme system of antioxidant protection in Black Sea mussels Mytilus galloprovincialis Lam. with pigmented and depigmented tissue structures. Dop. NNNU. 5: 162 – 166. Modern methods in biochemistry / edit. by V.N. Orekhovych. – М: «Meditsina», 1977. – p. 227-228.  Kiessling K. H., Lunquist C.G. (1962) Thiamin diphosphate in growind fissues. III. Pyruvate oxidation in liver mitochondria from young and from thiamine diphosphate deficient adult rats. Experim. Cell. Kes. 26: 189-197. Roche I.E. Cate R.J. (1977)  Purification of porcine liver pyruvate dehydrogenase complex and charactirization of its catalitic and regulatory properties.  Arch. of Biochim. and Biophys. 183: .664-677.                                                                                    

THE MECHANISMS INHIBITING PYRUVATE- AND OXOGLUTARATE DEHYDROGENASE COMPLEXES IN MUSSELS

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THE MECHANISMS INHIBITING PYRUVATE- AND OXOGLUTARATE DEHYDROGENASE COMPLEXES IN MUSSELS

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