Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor.

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.

Dissection of the humoral immune response toward an immunodominant epitope of HIV: a model for the analysis of antibody diversity in HIV+ individuals.

Understanding the dynamics of the humoral immune response to HIV epitopes in the presence of genetic drift and antigenic variation of the virus may reveal critical elements of protective immunity against HIV. Analysis of antibody maturation and diversity is difficult to study at the molecular level in humans. We used a combinatorial phage display peptide library to elucidate antibody diversity in HIV-infected individuals to a single immunodominant epitope in gp41. A serum sample derived from an HIV+ individual was used to screen a phage display a 12 mer cysteine-constrained loop peptide library. In doing so, we isolated mimotope-presenting phages corresponding to the immunodominant gp41 epitope CSGKLIC (residues 603-609). The mimotopes and control phages expressing epitope variants were reacted with a panel of 30 HIV+ sera. The patients showed distinct and variable recognition patterns compared with one another. Subfractions of the polyclonal sera were affinity purified and analyzed for epitope specificities. These analyses illustrated that epitope variants can be used to decipher antibody diversity. Elucidation of the plasticity of the humoral response and its polyclonality toward discrete epitopes contributes to our understanding of the antibody maturation process in individuals infected with viruses such as HIV.

Expansion of epitope cross-reactivity by anti-idiotype modulation of the primary humoral response.

The primary humoral response produces antigen-specific antibodies so to clear the initial infection, and generates a population of corresponding memory cells to prevent infection by future encounters with the same pathogen. The continuous genetic modification of a pathogen's exterior, however, is one mechanism used to evade the immune defenses of its host. Here we describe a novel means, involving anti-idiotypic antibodies, by which the host can counteract such pathogen genetic alterations by modulation of its primary humoral response. An autoimmune response against primary antibodies, Ab1's, creates anti-idiotypic antibodies (Ab2's), some of which (designated Ab2alpha) are able to bind the Ab1/antigen complex. We have discovered that binding of Ab2alpha to its corresponding Ab1 can expand Ab1's ability to bind variations of its antigen. This expanded epitope cross-reactivity is shown not only to increase the binding activity of Ab1 but also its ability to neutralize a variant infectious virus. MAb M77 is an Ab1, which is highly strain-specific for the HIV-1 envelope protein gp120(IIIB). This Ab1 can be rendered cross-reactive and neutralizing for an otherwise resistant HIV strain by its interaction with a unique anti-idiotypic Ab2alpha (GV12). Furthermore, molecular characterization of this expanded cross-reactivity was accomplished using combinatorial phage display peptide libraries.

Acetylcholine interactions with tryptophan-184 of the alpha-subunit of the nicotinic acetylcholine receptor revealed by transferred nuclear Overhauser effect.

Acetylcholine interactions with three genetically engineered fusion proteins containing peptides from the nicotinic acetylcholine receptor were studied by 1D and 2D nuclear magnetic resonance methods. The three proteins were Torpedo alpha 184-200, Torpedo alpha 186-198, and human alpha 183-204 of the acetylcholine receptor fused to the first 323 residues of the E. coli protein trpE. Nuclear Overhauser effect studies revealed interactions of bound acetylcholine with tryptophan-184 present in the Torpedo alpha 184-200, and the human alpha 183-204 sequences. These interactions are between the N(CH3)3+ and CH3 groups of acetylcholine with the aromatic protons of tryptophan. The appearance of these cross-peaks indicates a distance of less than 5 A between tryptophan and the bound ligand; however, direct contact has yet to be proven.

Identification of glycoproteins that are receptors for peanut agglutinin on immature (cortical) mouse thymocytes.

Binding of peanut agglutinin is being widely used as a marker for immature mouse thymocytes and for the separation of these cells from the mature thymocytes. Two cell surface glycoproteins that bind peanut agglutinin were detected on unfractionated as well as immature thymocytes by lectin overlay and affinity chromatography: one of Mr between 170 000 and 180 000, and the other, a minor component, of Mr 110000, both of which are partially sialylated. No receptors for peanut agglutinin were detected on the mature cells, whereas desialylation experiments revealed the presence of a glycoprotein of Mr 110000. These findings were corroborated by electrophoretic analysis of cell surface glycoproteins of the isolated thymocyte subpopulations labeled in their carbohydrate moieties.

Humoral immune response to immunocomplexed HIV envelope glycoprotein 120.

To further our understanding of the nature of HIV-1 immunogenicity, we injected mice with the virus envelope protein gp120 in different configurations: free, complexed with its receptor CD4, and as an immunocomplex with a monoclonal antibody directed against the V3 loop of the protein. Analyses of the polyclonal sera, as well as of monoclonal antibodies produced in each case, allowed us to conclude that the quality of the humoral immune response depended on the complexation state of the antigen. For the free gp120 and gp120-CD4 complex the responses were directed mainly toward conformational epitopes. However, gp120 immunocomplexed with anti-V3 loop Mab produced, in addition, numerous MAbs directed toward linear epitopes. Epitopes were mapped using immunoblots of gp120 cleaved with S. aureus V8 protease and a combinatorial epitope phage-display library. It was found that some of the linear epitopes had been previously identified as T cell epitopes. These results suggest that the immunocomplexed gp120 may be particularly well taken up by antigen-presenting cells, leading to the processing of the gp120 and the efficient presentation of T cell epitopes. Thus immunocomplexation should afford a means for enhancing the immunogenicity of gp120 and improving its presentation.

Alpha-bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots.

The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.

NMR studies of recombinant active site peptides of the nicotinic acetylcholine receptor.

Interactions of ligands with recombinant cholinergic binding sites have been monitored by NMR. Monitoring the selective T1 relaxation of the protons of acetylcholine, nicotine, d-tubocurarine, and gallamine reveals specific binding to peptide constructs containing the alpha 183-204 or shorter sequences of the nicotinic acetylcholine receptor of Torpedo, Human, Chicken, Xenopus, Mouse, Calf, and Drosophila. The trend of the KD values of the different ligands shows that the binding of the low molecular weight agonists and antagonists is very weak to the Drosophila sequence which is different from the vertebrate sequences in the N and C terminals. Within the vertebrates, the antagonists d-tubocurarine and gallamine display a KD trend different from that of acetylcholine and alpha-bungarotoxin. Specificity of binding is proven by the fact that atropine, a muscarinic inhibitor, binds non-specifically. Temperature dependence indicates a fast exchange limit (T1 bound greater than tau bound) for gallamine bound to the Torpedo alpha 184-200 sequence. This limit should apply also for the other ligands which have weaker binding constants.

Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots.

Three possible disulfides in the acetylcholine receptor alpha-subunit.

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.

Molecular decoys: ligand-binding recombinant proteins protect mice from curarimimetic neurotoxins.

Mimic ligand-binding sites of the nicotinic acetylcholine receptor bind d-tubocurarine and alpha-bungarotoxin in vitro. Injection of such binding sites into mice could act as molecular decoys in vivo, providing protection against toxic ligands. This hypothesis of molecular "decoyance" has been tested in greater than 250 mice. Bacterially produced cholinergic binding sites provided a 2-fold increase in the survival rate of animals challenged with curarimimetic neurotoxins. Possible considerations for decoy designs and their applications are discussed.

Comparison of the toxin binding sites of the nicotinic acetylcholine receptor from Drosophila to human.

Recombinant toxin binding proteins have been previously found to provide a convenient experimental system for the study of receptor-ligand recognition (Aronheim et al., 1988). Here, this system has been used to produce the binding sites of the cholinergic receptor derived from seven organisms, Torpedo californica, Xenopus, chick, mouse, calf, human, and Drosophila. These have been compared with respect to their toxin binding capacity. Scatchard analyses show that the KD values of alpha-bungarotoxin binding to the above sites are 63, 536, 150, 3200, 6200, 6470, and 1700 nM, respectively. These results reiterate the importance of alpha 183-204 as a ligand binding site. In order to increase the repertoire of sites available for study, chimeric structures were constructed. Through the analysis of such chimeras, some themes of the gross anatomy of the binding site can be learned. A positive subsite followed by a hydrophobic patch preceding a nucleophilic domain appears to be required for efficient toxin binding.