RESUMO
A method for fractionation of membrane structures of Yersinia pestis is developed. It involves the following basic stages: the cultivation of bacteria in a liquid nutrient medium, mechanical destruction from the solid state in the X-press or ultrasound treatment of the suspension, subsequent two-stage centrifugation in the step (70-15%) and linear (70-45%) gradients of the sucrose density, collection of fractions and their storage. The method makes it possible to separate rapidly and efficiently the outer and cytoplasmic membranes which preserve biochemical and morphological integrity. This is confirmed by the distribution pattern of marker enzyme activities, by the electron microscopic control as well as by other modern sediment tests. High heterogeneity of the polypeptide composition of the isolated membrane preparations has been shown by electrophoresis in PAAG in the presence of sodium dodecyl sulphate as well as definite sensitivity of certain protein subunits to variations of the temperature (28 or 37 degrees C) during cultivation of a plague agent.
