Differentiation isocitrate isoenzymes using DEAE-cellulose

Abstract

Study of physico-chemical and regulatory aspects of the isocitrate lyase is an urgent problem,
because at the moment there is little data on the subject. The aim of this work was to develop a multi-stage
purification scheme isocitrate lyase and its isoenzymes separation using ion-exchange chromatography on
DEAE-cellulose. For isocitrate lyase from various purification scheme was used objects, comprising
homogenization of the material, an ammonium sulfate fractionation 0-70% of saturation, gel-filtration on
Sephadex G-25 ion-exchange chromatography on a column of DEAE-cellulose.
Use of a four-step purification scheme yielded isocitrate lyase isoenzymes with high specific
activity. Thus, the shape of the corn ICL1 specific activity value was 2.85 U / mg protein and ICL2 - 3.1 U /
mg protein. A characteristic feature of the purification scheme used was the use of isocitrate ion-exchange
chromatography on DEAE-cellulose, and application of this step yielded two isoforms test preparations of
the enzyme from corn, soybeans and amaranth. Isozymes ICL stripped from the column of DEAE cellulose
with a gradient concentration of KCl. The degree of purification of homogeneous preparations ICL isoforms
ranged from 57 to 87 times. In our opinion, this was due to different values of the specific activity in the
homogenate on the test plants. It should be noted that the cleaning of the plant include isocitrate lyase
characteristic significantly higher yield of the enzymatic activity, which have ranged from 6.5 to 9% soybean
amaranth. The higher the output level of activity in the preparation of highly purified preparations DIC
appears to be explained by a significant content isocitrate lyase activity in plant facilities. Analysis of the data
indicates that as a result of four-step purification scheme obtained electrophoretically homogeneous isoforms
Rf = 0,28 and 0,44. Developed a multi-stage purification scheme involving ammonium sulfate salting out, gel
filtration on Sephadex G-25 and the use of ion exchange chromatography on DEAE-cellulose, which allowed
to obtain electrophoretically homogeneous preparations of isocitrate lyase isoenzymes of plants with different
types of basal metabolism. Preparation of highly purified isoenzymes ICL offers opportunities to study the
physico-chemical, catalytic and kinetic characteristics of the functioning of this enzyme system.