Our long-term objective is to gain an understanding of the role of the smooth endoplasmic reticulum (SER) a regulator of extracellular calcium, which acts as a messenger and modulator of numerous events following fertilization of the egg. Having isolated a pure preparation of vesicles from the SER of sea urchin eggs and characterized its calcium transport properties, we have now purified a protein from this organelle. This protein resembles calsequestrin from the sarcoplasmic reticulum in muscle, in the way it stains with Stains-All, in its mobility on polyacrilamide gels, in its behavior in the purification steps and in its calcium binding. Most importantly this protein cross reacts on Western blots with antibodies against cardiac calsequestrin. We have produced an antiserum against this calsequestrin-like protein from sea urchin eggs and one objective will be to determine its localization and distribution (presumably within the SER) in the unfertilized egg by immunolabelling using optical and electron microscopy. Its distribution will also be examined in the immature oocyte to assess changes associated with maturation and also at defined stages after fertilization. We will also attempt to correlate calcium release sites and the SER distribution following fertilization. Using immunotechniques, we will quantitate the protein in the oocyte, the egg and during early embryogenesis. Thus the antibody as a probe will shed light on possible changes in functional distribution of the SER associated with different functional states of the egg and embryo. We will also continue with further characterization of this purified protein from the SER of the sea urchin egg to determine whether it might serve a calcium """"""""storage"""""""" function in the SER of the sea urchin egg and whether it undergoes conformational changes in the presence of calcium. We will also determine if it interacts with other proteins in the egg. By establishing similarities and differences in the protein constituents of the SER and sarcoplasmic reticulum (a system which is reasonably understood) this research (and future studies on other constituents of the SER) will be important for further elucidation of the role of the SER in the regulation of intracellular calcium in the egg and early embryo. we will also continue with functional studies on an isolated vesicle preparation from the SER. Such information may, in the long run, be of value in understanding problems of infertility and birth defects in humans and also of abberations associated with cancer cell growth.
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