Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation.

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.

The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution.

The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.

Ribosome purification by isopycnic sedimentation in high density sucrose gradients.

The applicability has been investigated of a method for the isolation of cytoplasm (and mitochondrial) ribosomes of various eukaryotes. The procedure is based on centrifugation techniques and includes a sedimentation of the ribosomes in a sucrose gradient of very high density. It was found to be particularly useful with material which contains relatively few ribosomes.

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient.

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi-elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered ligh finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions. The application of the method to eukaryotic large ribosomal subunits is described as an example.

The molecular weight of Artemia ribosomes, as determined from their refractive-index increment and light-scattering intensity.

Cytoplasmic ribosomes were isolated from the cryptobiotic embryos of the brine shrimp Artemia salina. Measurements of their refractive-index increments and light-scattering intensities give a value for their molecular weight of (3.4+/-0.2)x10(6).

Physicochemical characterization of ribosomal particles from the eukaryote Artemia.

Cytoplasmic ribosomes and their large and small subunits have been isolated from the brine shrimp Artemia, and their basic physicochemical properties have been determined. From measurements of their ratio of absorbance at 260 and 280 nm, phosphorus and protein content, buoyant density in a CsCl gradient, and density increment, values have been derived for their RNA content (49, 52, and 43 +/- 2%, respectively), absorbance coefficient at 260 nm (12.1, 2.8, and 10.5, +/- 0.4 ml/mg, respectively), and partial specific volume (0.63, 0.62, and 0.65 +/- 0.002 ml/g, respectively). Analytical boundary sedimentation has yielded their standard sedimentation coefficient (81, 59, and 38, +/- 1 S, respectively). Photon correlation spectroscopy of laser light scattered from centrifuged solutions has yielded their translational diffusion coefficients (1.41 +/- 0.02, 1.61 +/- 0.03, and 1.80 +/- 0.04 X 10(-7) cm2/s, respectively). Combinations of these data have yielded values for their molecular weight (3.85 +/- 0.2, 2.45 +/- 0.2, and 1.45 +/- 0.1 X 10(6), respectively), for the total molecular weight of the free acid form of their RNA (1.9 +/- 0.1, 1.27 +/- 0.08, and 0.62 +/- 0.04 X 10(6), respectively) and of their proteins (2.0 +/- 0.1, 1.2 +/- 0.1, and 0.83 +/- 0.08 X 10(6), respectively), for their frictional coefficient ratio (1.54 +/- 0.04, 1.58 +/- 0.05, and 1.66 +/- 0.06, respectively), for their hydrodynamic and dry particle volume and radius, and for their hydrodynamic solvation. All the results are highly consistent. The various intact ribosomal particles of Artemia have nearly the same solvation (1.7 g/g); in this respect they resemble Escherichia coli ribosomal particles; this finding contrasts, however, with the few published data on eukaryotic ribosomes. This work strengthens the basis for a comparative study and for a more detailed elucidation of the structure of eukaryotic ribosomes.

Characterization of some ungrouped viruses.

Bangui, Bobaya, Kowanyama, Upolu and Zinga are enveloped spherical RNA viruses sensitive to ether and acid pH; morphologically resembling Bunyaviridae, their diameters range from 90 to 100 nm and their bouyant density is 1.17 to 1.18 g/ml in sucrose. St. Floris, a bunya-like virus, serologically related to Rift Valley Fever, has a diameter ranging from 90 to 110 nm. Triniti, Zingilamo, IPYM 120 and virus strain AnB4268 are enveloped spherical RNA viruses sensitive to ether and acid pH, have diameters of 65, 55, 55 and 60 nm and bouyant densities of 1.18, 1.24, 1.20 and 1.18 g/ml in sucrose, respectively; morphologically they resemble Togaviridae. Salanga is a poxvirus measuring 190 X 225 nm. Virus strain AnY1444 is a spherical, non-enveloped RNA virus measuring 85 nm, morphologically resembling Reoviridiae; it is restraint to both ether and acid pH and shows two-peak densities of 1.32 and 1.36 g/ml in caesium chloride. Bangoran, Keuraliba and Yata resemble Rhabdoviridae measuring 60 X 175, 65 X 195 and 60 X 185 nm, respectively.

High-pressure scattering study of Artemia salina ribosomes and polysomes.

The intensity has been measured of the light scattered by solutions of brine shrimp (Artemia salina) ribosomes and polysomes under hydrostatic pressures up to 2000 atm. This has given information about pressure-induced decreases in the means weight of the particles in solutions containing different concentrations of K+ and Mg2+ ions. The dissociation-association equilibrium reaction ribosome formed from large subunit + small subunit is accompanied by a volume change, --200 less than delta V less than --300 ml/mol; this delta V is discussed with relation to different models for the interaction between the ribosome subunits. The application of high pressures on polysome solutions caused also decreases of the light scattering; these were slower than in the case of ribosomes, and nonexponential. Only small decreases were found for ribosomes attached to messenger-RNA, which were obtained by incubation of polysomes with pancreatic RNAase. After fixation of the ribsomes and polysomes with formaldehyde, the light scattering remained constant with increasing pressures.

Regular, rodlike, macromolecular structures in Artemia salina.

Artemia salina of different origins and in different developmental stages contains regular, rodlike, macromolecular structures. The particles were visualized by electron microscopy after negative staining. They can be purified from the postmitochondrial supernatant by sucrose gradient centrifugation in the presence of EDTA. Their size distribution has been measured from electron micrographs. The particles are probably nucleoprotein complexes.

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.

Physical properties of Artemia salina ribosomes.

Eukaryotic ribosomes were isolated from the cryptobiotic embryos and from the further-developed free-swimming nauplii of the brine shrimp Artemia salina. Analytical boundary sedimentation and photon correlation spectroscopy yielded, respectively, the standard sedimentation and diffusion coefficients at infinite dilution, s degrees 20,w = 81 +/- 1 S and D degrees 20,w = (1.41 +/- 0.02) x 10(-7) cm2/s, for the unfixed and formaldehyde-fixed ribosomes from different developmental stages and for ribosomes attached to a messenger RNA fragment. Also, the density increment was determined, from which the partial specific volume was derived (0.63 +/- 0.01 cm3/g). Combination of the different measured parameters gives accurate values for the molecular weight (3.8 +/- 0.1) x 106 and for size and solvation parameters. These results are compared with their counterparts for the smaller ribosomes from the prokaryote Escherichia coli.