No abstract

The transforming protein of avian sarcoma virus UR2, p68v-s, has an associated tyrosine-specific protein kinase activity similar to that of p6O-SrC and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-s also is associated with phosphatidylinositol (PtdIns) kinase (22,23), tyrosine kinase activity shows little substrate specificity in vitro, and the phosphorylation stoichiometries observed are often too low to be of obvious regulatory significance (21). The possibility that the physiological substrate of the oncogene kinases might not be tyrosine was raised by recent observations that p6fV-src can phosphorylate glycerol (24,25). Because of (i) the similarities between the activity of oncogene kinases and that of growth factor receptors (16-18) and (ii) the implication of P-inositide turnover in cell proliferation, it occurred to us to test the phosphorylating activity of one of these kinases, p68vr's, towards phosphatidylinositol (PtdIns). The protein p68v-ros is the oncogene product of avian sarcoma virus UR2 (26, 27); although unrelated to the p60vsrc of Rous sarcoma virus (28), it will phosphorylate itself and exogenous protein substrates on tyrosine residues (27). We found that immunoprecipitated p68v-ros is also capable of phosphorylating PtdIns to Ptdlns 4-phosphate (PtdIns4P). Viruses and Antisera. The origins of UR2 and some of the properties of its transforming protein have been described (26,27). A temperature-sensitive (ts) mutant, UR2-R0200, has been isolated by one of us (P.B.). This mutant produces full expression of the transformed phenotype at 37°C but only partial expression at 42°C. Anti-gag antiserum was obtained from E. J. Smith.Cell Cultures and Conditions. Chicken embryo fibroblast cultures were prepared and maintained as described (26). Infected cells were passaged 2 or 3 times until extensive morphological conversion was apparent.Immunoprecipitation of p68vros. Cell lysates were prepared with either RIPA buffer or a buffer containing 1% NP-40 (or Triton X-100), 1% aprotinin, 10 mM Tris, 100 mM NaCl, and 1 mM EDTA (pH 7.4). Plates (10 cm) containing about 107 cells were washed three times in Tris/glucose, and

No abstract

Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 degrees C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 degrees C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 degrees C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS. Cels treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.

No abstract