The efficiency of cryoprotectors-polyethylene oxide 400, glycerol, oxyethylated glycerol, dimethyl sulphoxide was studied relative to stabilization isolated lysosomes under different regimes of freezing-thawing. It is established that a decrease in the amount of lysosome hydrolases (RNase, DNase, catepsin D, phosphatase) released to the environment is the most significant in the presence of the mentioned substances uncer slow freezing-thawing.
Possible usage of ESR probe method for studying low temperature effect on structural and functional state of mitochondria is under study. It is shown that during freezing out of mitochondrial water there is sharp dehydration of intermitochondrial matrix and membrane thickening. The facts are given about damage of barrier function of the membrane at the moment of appearance of extramitochondrial liquid phase during thawing.
Hybridization of M4- and H4-isoenzymes of lactate dehydrogenase in saline medium was studied during slow freezing down to -9.5; -23 and -30 degrees C. It is established that appearances of the enzyme hybrid forms is associated with free water freezing out and with an increase in the concentration of salts in the solution. No hybridization was observed under fast cooling of the enzyme down to -196 degrees C, under exposition in the concentrated solution of chloride and sodium phosphate without freezing and with freezing in deionized water. The mechanism of low-temperature hybridization of lactate dehydrogenase, based on concentration effects of salts which favour dissociation of a protein molecule into subunits, are under discussion. An assumption is advanced on an intensification of intermolecular interactions in liquid microphases of the frozen solution as a possible reason of the subunits recombination into hybrid isoenzymes.
The effect of various regimen of freezing and thawing on the functional state of one of the most important receptor-regulatory cell systems, i. e. adenylate cyclase complex (ATP-pyrophosphate lyase cyclating E C 4.6.1.1.), has been studied on isolated rat hepatocytes. Basal and fluoride-stimulating activity and enzyme susceptibility to the regulatory effect of isoproterenol, adrenaline and noradrenaline were shown. Regardless of the regimen used, freezing and thawing decrease the stimulating effect of adrenergetic agents on adenylate cyclase system of isolated hepatocytes. The reason of it is probably the damage of a receptor site. The functional properties of catalytic enzyme subunit practically do not change. The results obtained show the necessity of correction of metabolic cell responses mediated by adenylate cyclase system during their freezing and thawing.
