The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves


  • Andriy V. Semenikhin Separate division of the National University of Life and Environmental Sciences of Ukraine "Nizhyn Agricultural Institute", Ukraine
  • Volodymyr V. Sukhovieiev Nizhyn Mykola Gogol State University, Ukraine
  • Mykola V. Patyka National University of Life and Environmental Sciences of Ukraine, Ukraine
  • Vasyl S. Lukach Separate Division of the National University of Life and Environmental Sciences of Ukraine “Nizhyn Agricultural Institute”, Ukraine



chloroplasts, RuBisCO, ATP synthase, carbonic anhydrase, esterase activity, two-dimensional electrophoresis


Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.
Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton.
Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.
Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.


Junge, W.; Nelson, N. ATP Synthase. Annu. Rev. Biochem 2015, 84 (1), 631-657.

Malyan, A. N. Noncatalytic nucleotide binding sites: properties and mechanism of involvement in ATP synthase activity regulation. Biochemistry (Moscow) 2013, 78 (13), 1512 – 1523.

Schägger, H.; Cramer, W. A.; von Jagow, G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 1994, 217 (2), 220 – 230.

Srivastava, K.; Chaves, J. M.; Srivastava, O. P.; Kirk, M. Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses. Experimental eye research 2008, 87 (4), 356 – 66.

Wittig, I.; Karas, M.; Schägger, H. High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. Molecular & Cellular Proteomics 2007, 6 (7), 1215 – 1225.

Semenihin, A. V. Karboanhidrazna aktyvnist chynnyka spriazhennia CF1, izolovanoho z khloroplastiv shpynatu [Carbonic anhydrase activity of coupling factor CF1 isolated from spinach chloroplasts, in Ukrainian]. Reports of the National Academy of Sciences of Ukraine 2014, 9, 141 – 145.

Semenihin, A. V.; Zolotareva, O. K. Carbonic anhydrase activity of integral-functional complexes of thylakoid membranes of spinach chloroplasts. The Ukrainian Biochemical Journal 2015, 87 (3), 47 – 56.

Semenikhin, A. V.; Repanka, S. V.; Sukhovieiev, V. V.; Hurbuz, M. F. Metod dvokhvymirnoho elektroforezu u doslidzhenni polifermentnykh vlastyvostei ATF-syntazy khloroplastiv shpynatu. In Fundamentalni ta prykladni doslidzhennia v suchasnii khimii, Materialy V Mizhnarodnoi zaochnoi naukovo-praktychnoi konferentsii molodykh uchenykh [Method of two-dimensional electrophoresis in the study of polyenzyme properties of ATP synthase of spinach chloroplasts. In Basic and applied research in modern chemistry, Proceedings of the V International Correspondence Scientific and Practical Conference of Young Scientists], Nizhyn, Apr 12, 2018; Sukhovieieva, V. V., Ed.: Nizhyn Mykola Gogol State University: Nizhyn, 2018; 119 – 123 (in Ukrainian).

Lowry, O.; Rosebrough, N.; Farr, A. L.; Randall, R. PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENT. J. Biol. Chem. 1951, 193 (1), 265 – 275.

Andersson, L.-O.; Borg, H.; Mikaelsson, M. Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett. 1972, 20 (2), 199 – 202.

Kolesnichenko, A. V.; Ostroumova, E. A.; Zykova, V. V.; Vojnikov, V. K. Belki chetyrekh vidov zlakov, immunohimicheski rodstvennye stressovomu belku 310 kD. Fiziologiya rastenij [Proteins of four types of cereals, immunochemically related to the stress protein 310 kD. Plants physiology] 2000, 47 (2), 199 – 202 (in Russian).

Allen, J. M.; Hyncik, G. Localization of alkaline phosphatases in gel matrices following electrophoresis. Journal of Histochemistry & Cytochemistry 1963, 11 (2), 169 – 175.

Gomori, G. Preparation of buffers for use in enzyme studies. In Methods Enzymol., Academic Press: 1955; Vol. 1, pp 138 – 146.

Ignatova, L. K.; Rudenko, N. N.; Mudrik, V. A.; Fedorchuk, T. P.; Ivanov, B. N. Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII. Photosynth Res 2011, 110 (2), 89 – 98.

Wynne, D.; Ginsburg, S.; Shalitin, Y. Beef liver esterase: II. Kinetic properties. Arch. Biochem. Biophys. 1973, 154 (1), 204 – 211.

Lowry, O. H.; Lopez, J. A. The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem. 1946, 162 (3), 421 – 428.

Nikulina, G. I. Obzor metodov kolorimetricheskogo opredeleniya fosfora po obrazovaniyu molibdenovoj sini [Review of methods for the colorimetric determination of phosphorus by the formation of molybdenum blue]; Nauka: Moscow-Leningrad, 1965 (in Russian).



How to Cite

Semenikhin, A. V.; Sukhovieiev, V. V.; Patyka, M. V.; Lukach, V. S. The Effect of Exogenous Factors on the Polyenzymatic Activity of RuBisCO and ATP Synthase of Chloroplasts from Pea Leaves. J. Org. Pharm. Chem. 2021, 19, 21-27.



Original researches