Metalloproteinases (MMP) are proteolytic enzymes whose activity is determined by the presence of zinc ion in the active site and are a widely distributed family of protease to ensure the preservation of homeostasis in animals. In human body they also play many important functions participating in the process of embryogenesis as well as in the formation of blood cells or bone formation. The main role, as it seemed at the beginning research on this group of enzymes, was to digest the extracellular matrix. However, over the years, it turned out that metalloproteinases are important part in the regulation of cell biology, in particular a tumor cell. Studies have confirmed the participation of metalloproteinases in all stages of the process of carcinogenesis. At the moment, there are numerous reports of available of various MMPs in different tumors. However, two of these MMP–2 and MMP–9 belonging to the gelatinases seem to be the most common. It has been confirmed also that higher levels usually indicate a worse prognosis for the patient. Therefore, they may prove to be a valuable prognostic indication for clinicians. Attention was paid to the potential use of matrix metalloproteinases in targeted therapy. The pharmaceutical industry has long been trying to launch metalloproteinase inhibitors as possible therapeutics in cancer, seeing in them a potential that, however, is limited to the early stages of development. After initial setbacks related primarily to the low selectivity is used at once more modern methods as monoclonal antibodies or liposomal drug delivery system. Thus, giving the possibility of treatment that does not cause so enormous side effects as conventional therapy.
Introduction: Cysteine proteases are enzymes that regulate numerous physiological and pathological processes in the human body. Disorders of their activity can lead to a number of diseases. They play an important role in the process of carcinogenesis, participating in the invasion, transformation, angiogenesis, apoptosis and metastasis. The aim of this study was to elaborate the electrophoretic method of cysteine proteinases identification in the sera of patients suffering from chronic lymphocytic leukemia based on biotinylated iodoacetamide. Material and methods: Preliminary studies were carried out on the commercially available papain (EC 3.4.22.2) well known and widely used plant cysteine protease with a molecular weight 23,4 kDa. The study was conducted on the blood samples taken from patients with chronic lymphocytic leukemia (CLL) and control sera from healthy donors. The sera after the preincubation with iodoacetamide were mixed with the sample buffer followed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate (SDS–PAGE). The separated proteins were electrophoretically transferred to the nitrocellulose membranes and subjected to the further analysis using streptavidin conjugated with horseradish peroxidase (HRP). The use of substrate for HRP 3,3’- diaminobenzidine tetrachloride (DAB) allows the biotinylated iodoacetamide and thereby cysteine proteinase identification. Results: The comparative analysis of the sera from the patients with chronic lymphocytic leukemia and the control sera led to the identification of additional protein with a cysteine protease characteristic having a molecular weight of about 37 kDa, which did not occur or was present in a smaller amount of the control sera. Conclusions: The developed method allows the detection of cysteine proteases which are present in the control sera and the sera of patients with chronic lymphocytic leukemia.
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