T-kinase was extracted from the tissues of brain and liver, from plants and bacteria. In literature there are only few works where T-kinase purification to homogenous state from yeasts, higher plants and rats liver is described. Determination of T-kinase activity in organism tissues at different pathological states is insufficiently presented in literature and there are only several works on study of the enzyme activity in the liver of animals with tumors.
The aim of the study was to elaborate a technique of preparative extraction and partial purification of T-kinase for subsequent study of its properties.
Gradual fractional sedimentation was carried out by ammonium sulfate when saturating protein solution up to 20, 40, 60 and 80%. Each fraction was dialyzed in analogous conditions. Determination of free and total thiamine in dialyzates was carried out using tiochrome method by G. D. Yeliseyeva.
At analysis of myometrium tissue with tumor there was shown loss of T-kinase activity in the process of dialysis and step-by-step fractionation: protein solutions after dialysis, saturated by ammonium sulfate (at the concentrations 20% and 80%) did not possess enzymatic activity. At 40% saturation by ammonium sulfate of protein solution of myometrium tissue with benign neoplasm and at 60% saturation by ammonium sulfate of protein solution of myometrium tissue with malignancy one fraction of T-kinase appeared.
Conclusions:
1. The method of step-by-step fractionation facilitates T-kinase immobilization that results in conformational changes which enhance activity and stability of the enzyme.
2. Malignization process leads to narrowing of multiple spectrum of the enzyme fractions: from three fractions in non-malignant tissue to one in myometrium tissue with benign neoplasm and with malignancy.