Ion-Exchange Chromatography-Based Purification and Characterisation of Isocitrate Lyase Isoforms from Corn Scutella: Insights into Enzyme Function
Saba Hadi *
Biology Department, College of Science, Mustansiriyah University, Baghdad 10001, Iraq.
Zahraa B. Mohammed
Department of Biology, College of Education for Women, University of Kirkuk, Kirkuk, Iraq.
Huda F. Ramadan
Department of Biology, College of Education for Women, University of Kirkuk, Kirkuk, Iraq.
*Author to whom correspondence should be addressed.
Abstract
Background: Isocitrate lyase is widespread in bacteria, mushrooms and higher plants. In metazoans, the enzyme is present in some nematodes during the adult stage, post- embryonic larvae and embryos and probably in some arthropods. The presence of ICL and the glyoxylate cycle in vertebrates remains debated.
Aim: The purpose of this study is to devise a method for separating isocitrate lyase isoenzymes from corn scutella by using ion-exchange chromatography.
Methods: The isocitrate lyase enzyme was purified from corn plants using ion exchange chromatography and sulfate precipitation techniques. Enzyme activity was assessed using spectrophotometric methods, and the molecular weight was determined through gel chromatography. Studies were conducted to investigate the influence of pH, glycine, and glycolate on enzyme activity. The specific identification of the enzyme was determined using a modified Schiff reagent. SDS- PAGE electrophoresis was performed with a 12.5% polyacrylamide gel concentration. Standard marker proteins (Sigma) were used to plot the calibration curve.
Results: Two isoforms of isocitrate lyase, ICL1 and ICL2, were purified, exhibiting molecular masses of 164 kDa and 208 kDa, respectively. ICL1 demonstrated optimum activity at pH 7.5, while ICL2 exhibited optimum activity at pH 6. In this study, specific concentrations of glycine and glycolate were found to enhance the enzymatic activities of both isoforms. A highly statistically significant result was obtained using the P – value of less than.05. Post-hoc Tukey's test showed that the specific activity of ICL2 was significantly higher than that of ICL1, and the Km value of ICL2 was significantly greater than that of ICL1 (P < .05). These results further support the hypothesis that the two isoforms of ICL have distinct functional roles in metabolic processes.
Conclusion: This research provides significant insights into the characteristics of the isocitrate lyase enzyme in corn plants. The data indicate the presence of distinct enzyme forms with specific interactions in varying environmental conditions, which may be applicable in agricultural practices to increase crop yield and improve the metabolic turnover of organic acids.
Keywords: Isocitrate lyase isoenzymes, corn scutella, chromatography, glycine, glycolate