Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between the oligomycin sensitivity conferring protein and the F0 sector of the mitochondrial adenosinetriphosphatase complex: cooperative effect of the F1 sector

Beef heart mitchondrial oligomycin sensitivity conferring protein (OSCP) labeled with [14C]-N-ethylmaleimide ([14C]OSCP) at the only cysteine residue, Cys-118, present in the sequence [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I.V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22] exhibits full biological activity in a reconstituted F0-F1 system [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. The binding parameters of [14C]OSCP with respect to the F0 sector of submitochondrial particles largely depleted of F1 and OSCP (AUA particles) have been explored. In the absence of added F1, a limited number of high-affinity OSCP binding sites were detected in the AUA particles (20-40 pmol/mg of particles); under these conditions, the low-affinity binding sites for OSCP were essentially not saturable. Addition of F1 to the particles promoted high-affinity binding for OSCP, with an apparent Kd of 5 nM, a value 16 times lower than the Kd relative to the binding of OSCP to F1 in the absence of particles. Saturation of the F1 and OSCP binding sites of AUA particles was attained with about 200 pmol of both F1 and OSCP added per milligram of particles. The oligomycin-dependent inhibition of F1-ATPase bound to AUA particles was assayed as a function of bound OSCP. At subsaturating concentrations of F1, the dose-effect curves were rectilinear until inhibition of ATPase activity by oligomycin was virtually complete, and maximal inhibition was obtained for an OSCP to F1 ratio of 1 (mol/mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.     

Leukotriene B3, leukotriene B4 and leukotriene B5; binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Influence of Temperature Stress on in Vitro Fertilization and Heat Shock Protein Synthesis in Maize (Zea mays L.) Reproductive Tissues

This study was conducted to investigate the response of maize (Zea mays) male and female mature reproductive tissues to temperature stress. We have tested the fertilization abilities of the stressed spikelets and pollen using in vitro pollination-fertilization to determine their respective tolerance to stress. The synthesis of heat shock proteins (HSPs) was also analyzed in male and female tissues using electrophoresis of ³⁵S-labeled proteins and fluorography, to establish a relationship between the physiological and molecular responses. Pollen, spikelets, and pollinated spikelets were exposed to selected temperatures (4, 28, 32, 36, or 40°C) and tested using an in vitro fertilization system. The fertilization rate is highly reduced when pollinated spikelets are exposed to temperatures over 36°C. When pollen and spikelets are exposed separately to temperature stress, the female tissues appear resistant to 4 hours of cold stress (4°C) or heat stress (40°C). Under heat shock conditions, the synthesis of a typical set of HSPs is induced in the female tissues. In contrast, the mature pollen is sensitive to heat stress and is responsible for the failure of fertilization at high temperatures. At the molecular level, no heat shock response is detected in the mature pollen.     

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.     

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.