ISOLATION OF MATRIX METALLOPROTEINASE-2 FROM NONMALIGNANT BR EAST TISSUES IN WOMEN

Abstract

Matrix metalloproteinase - 2 (ММР-2) is zinc- and calcium-dependent extracellular proteinase which takes part in reorganizations of extracellular matrix in many physiological and pathological processes. The research aim is to develop the method of ММP-2 isolation from the nonmalignant tissue mammary gland in women. To obtain MMP-2 from breast tissues a modified method of Cawston and Tyler (1979) was used. Modification consisted in gradual precipitation of (NH4)2SO4 and application of Zn2+ and Са2+ ions for activation and stabilization of enzyme activity. Sampling of anatomical materials for research was conducted with compliance of ethical and legal standards. The homogenates of the resected tissues were dialyzed against the distilled water without salts and also with 2.0 mM solutions of ZnCl2, CaCl2 or mixtures of these salts. Dialysates were subjected to step-by-step fractionating (besieging at 20, 40, 60 and 80 % saturation) of (NH4)2SO4 with subsequent centrifugation at 9000g for 45 minutes. Resulting fractions were subjected to the repeated dialysis under the same conditions to remove the excess of (NH4)2SO4. Activity of ММP-2 was detected by hydrolysis of 0.001 % gelatin solution, the protein content – by the Loury method. The maximal specific activity of ММP-2 was determined in the fraction obtained at 60 % saturation by (NH4)2SO4 in presence of mixture of Zn2+ and Са2+ ions. The highest purification of ММP-2 (231.77 times) and maximal percentage of enzyme yield (349.41 %) were obtained in fraction with 60 % (NH4)2SO4 saturation in presence of Са2+ ions. The substantial increase of purification coefficient during fractionating by (NH4)2SO4 in presence Zn2+ and Са2+ ions testifies to the necessity of adding these ions for activation and stabilization of the enzyme structure. The results of the research indicate that the step-by-step sedimentation by (NH4)2SO4 leads to the fractionation of ММP-2 of untransformed female breast tissues at 60-80 % (NH4)2SO4 saturation (85.0 % of the entire activity). Presence of proteinase activity in the fractions obtained at other (NH4)2SO4 saturations, that is only 15.0 % of the total activity of enzyme, testifies, probably, to presence of multiple forms of ММP-2.