G. Werz

Changes in frequency and total number of nuclear pores in the life cycle of Acetabularia.

Abstract The frequence and the total number of nuclear pores were determined in electron micrographs of frozen-cleaved nuclei in various stages of the life cycle of the green algae Acetabularia mediterranea and A. (Polyphysa) cliftonii . In the course of the life cycle of the two species marked changes of both values were noted. Minimal pore densities and pore numbers occur in the gamete cell nucleus (about 8–10 pores/μm 2 and about 90–280 pores/nucleus), whereas maximal values are found in the mature primary nucleus of the vegetative phase (about 60–90 pores/μm 2 and about 4.9 – 7.5 million pores/nucleus). The data are discussed in relation to the transcriptional activities of the corresponding nuclear stage.

Cytoskeletal elements and their involvement in Polyphysa (acetabularia) protoplast differentiation: Cytoskeleton modifiers and concanavalin A-mediated effects

Abstract Protoplasts from coenocytic cap rays of the marine green alga Polyphysa (Acetabularia) cliftonii were treated with microtubule- (colchicine) and microfilament modifiers (cytochalasin A and B), to examine the involvement of cytoskeletal elements in gametangial morphogenesis as well as the presence or absence of a cytoskeletal transmembrane control of concanavalin A (ConA) receptors. The antitubulin inhibits mitosis, disrupts the anchorage of the nuclei and prevents the demarcation of the lid in the gametangial wall, but induces fluorescence-detectable ‘pre-lids’. It does not interfere with the partitioning of coenocytic protoplasts into uninucleate protoplasts, with the development of gametangial shape, and with the initiation of cellulosic walls. Cytochalasins suppress protoplasmic cleavage, reduce mitotic division and lead to the occurrence of ‘twin’ nuclei. They do not prevent cellulosic wall formation and the expression of lids. The combined effects of either colchicine or cytochalasins with ConA are cumulative. It is concluded that microtubules or microfilaments are not directly involved in cell wall initiation and synthesis, and furthermore, exert no direct transmembrane control on the exposure of ConA receptors, or ConA receptor binding.

Concanavalin A affects polysaccharidic wall formation and mitotic activity in Polyphysa (Acetabularia) cliftonii protoplasts

Abstract Cell wall formation, terminating the differentiation of protoplasts from nucleate cap ray cytoplasm of Polyphysa (Acetabularia) cliftonii, is inhibited by concanavalin A (ConA), depending on its concentration. The effect is completely reversible by application of the hapten sugar αMM, but not by NAcGal. It is concluded that ConA binds to certain glycosylated surface components, thus preventing the growth of the wall-polysaccharide chains, predominantly cellulose, or their attachment to ‘priming’ components. Normally, cell wall formation and nuclear divisions are strictly correlated. ConA, however, dissociates cell wall formation from the mitotic events reversibly in that it suppresses wall formation and leaves mitosis unmolested. In general, therefore, ConA revealed no mitogenic effect in the plant system under investigation. In special, the ConA effect might be regarded as a strong stimulation of mitotic events in relation to the normally existing correlation between mitosis and the presence of cell wall.

Small-angle X-ray scattering of D-Ribulose-1,5-diphosphate carboxylase from Dasycladus clavaeformis roth (Ag.) in solution

Abstract D-Ribulose-1,5-diphosphate carboxylase from Dasycladus was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of R g = 45.5 A was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 A and inner radius of 12 A. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density.

Electron microscopic studies on microcrystals of D-ribulose-1, 5-biphosphate carboxylase from Dasycladus clavaeformis Roth (Ag.).

Abstract Crystals of D-ribulose-1,5-biphosphate carboxylase (E.C. 4.1.1.39), naturally occurring in the extraplasmatic space of the unicellular green algae Dasycladus clavaeformis (Dasycladaceae), were studied by means of electron microscopy and optical diffraction. Optical diffraction patterns were obtained from thin sections. It is shown that the crystals are composed of cubic unit cells with α ∼ 31.5 nm. The density of the crystals was estimated as 1.07 ± 0.005 g/ml, a value that gives evidence of the presence of 12 enzyme molecules per unit cell. Optical diffraction studies of the thin sectioned crystals revealed 4mm -symmetry with four 2-fold rotation axes, resulting in at least a 222-symmetry.

On the spatial structure of a plant cell wall protein. Secondary structure of a cell wall protein fromAcetabularia

SummaryThe structure of a purified protein associated with the cell wall polysaccharides of the marine green algaeAcetabularia (Polyphysa) cliftonii has been studied by means of X-ray diffraction, infrared spectroscopy and circular dichroism. The homogeneous preparation of the cell wall protein has a molecular weight of 14,000, as determined by sodium-dodecylsulfate electrophoresis. Regular layer line reflections on the X-ray diffraction photographs suggest that a distinct order exists in the arrangement of the protein fibrils. Through infrared spectroscopy of thin aqueous films of the protein, as well as of the fibers, it was established that the α-helical structure is predominant in the cell wall protein. The fibers crystallize in a hexagonal unit cell witha=14.5 Å and c=27.0 Å, at a water content of two molecules per residue. Increase in water content causes an increase in thea-axis, but without change in thec-direction, thus keeping the α-helical conformation. Moreover the spectral data in the amide A, I, II, III, and IV-regions show that the cell wall protein has an ordered α-helical conformation.

Cell wall polypeptides of Polyphysa (Acetabularia) cliftonii: amino acid composition of stalk and cap cell wall polypeptides.

The amino acid composition of stalk and cap cell wall polypeptides of the unicellular alga Polyphysa (A.) cliftonii has been investigated. In spite of chemical and physical differences between stalk and can cell wall polysaccharides, the amino acid composition of the cell wall polypeptides appeared qualitatively similar in both structures. however, quantitative differences have been observed. The results are discussed on the basis of a possible role of the polypeptides in the growth of the cell wall.

Solution properties of a cell wall protein fromAcetabularia (Polyphysa) cliftonii

SummaryStudies have been performed on the precipitation of a cell wall protein, isolated from the marine green algaAcetabularia, from dilute aqueous solutions over the pH range where the α-helical conformation is maintained. The major purpose of this study was to establish the molecular conformation of a naturally occurring polypeptide of a molecular weight of 14,000 in the precipitate and to outline the mechanism of the precipitation. Since the precipitation behaviour from homogeneous protein solutions is important in relation to solution properties of the cognate polysaccharide chain from this alga, it was necessary to investigate the different conformations of the cell wall protein, including the possible aggregational states. Utilizing molecular weight fractions it can be shown that there are two distinctly different precipitation regions that depend on temperature, concentration of the protein, and pH. In one of these regions, the so-called α-region, precipitation occurs only in the α-helical conformation, without any conformational change if the physical conditions are varied. The temperature coefficient of the precipitation process in this α-region indicates that it must be nucleation controlled.

Evidence for a folded conformation of the cell wall protein fromAcetabularia (Polyphysa) cliftonii

SummaryThe cell wall protein fromAcetabularia has a non-random structure in aqueous solution at pH 5.3, as determined on the basis of intrinsic viscosity, sedimentation velocity and small angle X-ray scattering experiments. This non-random structure is stable in a pH range of 4.5–6.8, as observed on the basis of circular dichroism and viscosity measurements, supporting that the cell wall protein has a specific folded structure. All hydrodynamic measurements, including small angle X-ray scattering in solution, in this pH range are consistent with a prolate ellipsoid model for the shape of this protein, with overall dimensions ofc=86.0 Å,b=7.0 Å, anda=7.5 Å, and with a radius of gyration ofR=39.5 Å. The possibility of a coiled shape was investigated using a worm-like chain model, but it was inconsistent with the experimental data. Instead, a filled particle with uniform density which is equivalent in the scattering behavior is proposed. By a comparison of the observed radius of gyration, Rg=39.5 Å, and the radius of gyration of the cross section,Rc=7.5 Å, we were able to describe the cell wall protein in terms of a prolate ellipsoid of revolution. Comparisons of the experimental scattering curve, plotted as logl (h) versus logh, with the corresponding plots of normalized intensities, calculated for particles of particular shape and various axial ratios indicate a very asymmetric shape for the cell wall protein fromAcetabularia.