Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes.

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.

Characterization of the Strongylocentrotus purpuratus egg cell surface receptor for sperm.

In earlier studies from our laboratory, the intact sperm receptor was partially purified from Strongylocentrotus purpuratus crude egg membranes, but due to its insolubility, it was not possible to purify it to homogeneity. Nonetheless, this receptor preparation bound with species specificity to acrosome-reacted sperm, thereby inhibiting fertilization. Antibodies against the partially pure receptor inhibited fertilization in S. purpuratus (but not Arbacia punctulata) by coating the egg surface, indicating the presence of binding sites that can be species-specifically recognized by both sperm and antibody molecules. Recently we were able to further purify and characterize the receptor from S. purpuratus eggs. Chaotropic agent solubilization of the receptor prepared from crude egg membranes yielded a very high molecular weight glycoconjugate that had many of the properties of a proteoglycan. The receptor interacted with binding in an in vitro assay and bound with species specificity to acrosome-reacted sperm to inhibit fertilization. Unfortunately, this receptor preparation was soluble only in certain chaotropic agents. Exhaustive Pronase digestion of the intact receptor yielded a soluble high-molecular-weight (greater than 10(6)) polysaccharide that was virtually devoid of protein. This glycosaminoglycan-like fragment was highly sulfated, and contained fucose, galactosamine, and iduronic acid. The fragment inhibited fertilization, but did not do so with species specificity. Recently, soluble molecules with receptor activity were generated by treating intact dejellied eggs with trypsin. These proteolytically derived molecules contained (on a weight basis) approximately equal amounts of protein and carbohydrate. Importantly, they inhibited fertilization with species specificity. The results suggested that the binding activity was conferred by the polysaccharide component of the receptor and that the intact receptor and the tryptic fragments contained structural elements in the polypeptide chain necessary for species recognition.

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells.

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.