Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate

The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for these detergents [Lichtenberg, D., Robson, R.J., & Dennis, E.A.(1983) Biochim. Biophys. Acta 737, 285-304]. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At that point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations (from 0.25 to 20 mM phospholipid). The results allowed a quantitative determination of RSat, the effective detergent to lipid molar ratios in the saturated liposomes, which were 0.64, 1.3, and 0.30 for Triton X-100, octyl glucoside, and sodium cholate, respectively. The corresponding ratios in the mixed micelles, RSol, were 2.5, 3.8, and 0.9 mol of detergent/mol of phospholipid. The monomer concentrations of the three detergents in the aqueous phase were also determined at the lamellar to micellar transitions (0.18, 17, and 2.8 mM, respectively). These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparations. A rough approximation of the partition coefficients was derived from the turbidity and permeability data (K = 3.5, 0.09, and 0.11 mM-1 for Triton X-100, octyl glucoside, and sodium cholate, respectively). It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents.     

Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).     

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.     

Polyamine stimulation of protein phosphorylation in isolated pea nuclei

The phosphorylation of several proteins in isolated nuclei from Pisum sativum L. was stimulated by spermine. Although spermine increased the general protein phosphorylation by 10 to 20%, it increased the phosphorylation of a 47 kilodalton polypeptide by 150%. By comparison other polyamines, spermidine, putrescine, and cadavarine had far less effect on the phosphorylation of the 47 kilodalton or any other polypeptide. Sodium fluoride was able to inhibit the phosphorylation of the 47 kilodalton polypeptide in the control, implying the participation of protein phosphatase(s) in the phosphorylation of nuclear proteins. Spermine stimulated the phosphorylation of the 47 kilodalton polypeptide over the controls, even in the presence of NaF. This result indicates that spermine probably activates a nuclear kinase, a conclusion supported also by thiophosphorylation data. The inability of ethyleneglycol-bis (β-amino-ethyl ether)-N, N′-tetraacetic acid and Compound 48/80, a calmodulin antagonist, to inhibit this spermine stimulated phosphorylation renders improbable any role of calcium and calmodulin in mediating this response.     

Characterization of monoclonal antibodies to oat phytochrome by competitive radioimmunoassays and comparative immunoblots of phytochrome peptides

A library of 50 hybridomas which make antibodies to oat phytochrome was produced from the fusion of spleen cells from immunized Balb/c mice with P3x63Ag8 myeloma cells. Hybridomas were selected in a medium containing hypoxanthine, aminopterin, and thymidine, and specific hybridomas were screened for production of antibodies to phytochrome using a solid-phase enzyme-linked immunoadsorbent assay which was antigen specific. Positive cultures were cloned three times by limit dilution to assure monoclonal growth and stability. Specificity toward phytochrome was established by Western blot analysis and immunoprecipitation. Epitope specificity of nine monoclonal antibodies was determined by competitive inhibition radioimmunoassays and/or comparative immunoblots of tryptic peptides of phytochrome.     

Antibodies to Z DNA stabilized with polyarginine

The left-handed form of poly(dG-m5dC).poly(dG-m5dC) induced by heating the copolymer in the presence of magnesium and stabilized with polyarginine can be used to raise antibodies in rabbits. These antibodies are able to recognize the Z conformation of both methylated and nonmethylated forms of the copolymers. In the same experimental conditions, hypermethylated B DNA is not recognized by these antibodies.     

Cellular and subcellular localization of calcium in gravistimulated corn roots

Summary Antimonate staining procedures and energy dispersive X-ray microanalytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.