Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

Ribonucleic acid and protein content of eukaryotic ribosomes isolated from Artemia salina

We are studying the ribosomes from the cryptobiotic embryos of Artemia salina. Here we report on the relation between the optical density at 260 nm of a ribosome solution and its RNA and protein content. Using the original Lowry method or a modified version, it has been found that 1 A₂₆₀ unit contains 42.4 ± 1.6 μg of protein, and, from the phosphorus content, that the same solution contains 41.6 ± 1.0 μg of RNA. Analytical isodensity equilibrium centrifugation gives a value of 1.570 ± 0.005 g/cm³ for the buoyant density of these ribosomes in CsCl. This density can be related to a protein content of 51%, which is in accord with the chemical determinations. The relation between the optical density of ribosomes, RNA, and protein content and the optical density of rRNA of different systems, such as Escherichia coli, yeast, A. salina, and rat liver is discussed.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Elongation factor 2 from Artemia salina embryos and its affinity for ribosomes

Crude extracts from Artemia salina undeveloped embryos do not contain detectable elongation-factor-2 (EF2) kinase and endogenous ADP-ribosylating activities. Accordingly, EF2 purified from this source is an enzyme relatively free from phosphorylated and ADP-ribosylated forms. Endogenous ADP-ribosyltransferase activity appears only after purification of EF2. The affinities of EF2 and of ADP-ribosyl-EF2 for ribosomes from A. salina undeveloped embryos have been calculated by measuring the ability of the factors to inhibit the N-glycosidase activity of ricin on ribosomes.     

Diffusive properties of water in Artemia cysts as determined from quasi-elastic neutron scattering spectra.

Results have been obtained on the quasi-elastic spectra of neutrons scattered from pure water, a 20% agarose gel (hydration four grams H2O per gram of dry solid) and cysts of the brine shrimp Artemia for hydrations between 0.10 and 1.2 grams H2O per gram of dry solids. The spectra were interpreted using a two-component model that included contributions from the covalently bonded protons and the hydration water, and a mobile water fraction. The mobile fraction was described by a jump-diffusion correlation function for the translation motion and a simple diffusive orientational correlation function. The results for the line widths gamma (Q2) for pure water were in good agreement with previous measurements. The agarose results were consistent with NMR measurements that show a slightly reduced translational diffusion for the mobile water fraction. The Artemia results show that the translational diffusion coefficient of the mobile water fraction was greatly reduced from that of pure water. The line width was determined mainly by the rotational motion, which was also substantially reduced from the pure water value as determined from dielectric relaxation studies. The translational and rotational diffusion parameters were consistent with the NMR measurements of diffusion and relaxation. Values for the hydration fraction and the mean square thermal displacement [u2] as determined from the Q-dependence of the line areas were also obtained.     

The high molecular weight and the low molecular weight acid phosphatases of the frog liver and their phosphotyrosine activity

1. Two molecular weight classes of non-specific acid phosphatases (AcPases) (3.1.3.2) are present in the frog (Rana esculenta) liver: a higher molecular weight (HMW) of Mr 140,560 and a lower molecular weight (LMW) of Mr 38,180 enzyme. 2. The LMW AcPase was described earlier and the HMW AcPase of optimum pH 4.8 is shown to be a L(+)-tartrate sensitive, thermolabile, dimeric glycoenzyme slightly activated by DTT. 3. The HMW and the LMW AcPases exhibit activity for phosphotyrosine which showed similar sensitivity to various effectors as the p-nitrophenyl phosphatase activity; however, both enzymes differed substantially in this respect suggesting that they might be involved in different metabolic steps.     

The molecular weight cut-off of microcapsules is determined by the reaction between alginate and polylysine

Mammalian cells encapsulated in alginate-polylysine microcapsules are used as artificial organs in cancer research and in biotechnology. These applications require microcapsules with a reproducible mol. wt. cut-off. The high cost of the polycation, polylysine, requires an efficient preparation procedure. This article shows that the overall reported contact time of 5 minutes at ambient conditions should be increased several times in order to reach a maximal binding between the calcium alginate beads and 0.1% (w/v) polylysine solutions. An increase of the polylysine concentration from 0.0125% to 0.8% (w/v) resulted in a faster maximal binding, but the amount of polylysine bound increased also. Immersion of calcium alginate beads with a diameter of 750 mum, prepared from 1 mL alginate, in 30 mL of a 0.8% (w/v) polylysine solution, resulted in a polylysine spill of more than 89%. The time required to reach a maximal binding was related to the reaction temperature. The interaction zone between calcium alginate beads and fluorescein isothiocyanate-labeled polylysine solutions was visualized with a confocal laser scanning microscope as a function of time. Microcapsules, prepared at 40 degrees C with 0.1% (w/v) polylysine solutions with mol. wts. between 12 and 249.2 kD, were permeable for fluorescein isothiocyanate-labeled dextran, mol. wt. 4.7, but not for 40.5 kD. Higher polylysine concentrations resulted in a membrane with a mol. wt. cut-off lower than 4.7 kD. (c) 1993 John Wiley & Sons, Inc.     

Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA

Synaptophysin is a major glycoprotein of Mr approximately 38,000 (in deglycosylated form: Mr approximately 34,000) characteristic of a certain class of small (30-80 nm diameter) neurosecretory vesicles, including presynaptic vesicles, but also vesicles of various neuroendocrine cells of both neuronal and epithelial phenotype. Using synaptophysin-specific antibodies we have isolated cDNA clones from rat nervous tissue libraries, which identify an approximately 2.5-kb mRNA in rat and human cells, including neuroendocrine tumours, that contains a reading frame for a polypeptide of 307 amino acids with a total mol. wt of 33 312. The deduced amino acid sequence, which was partly confirmed by comparison with sequences of two tryptic peptides obtained from purified synaptophysin, revealed four hydrophobic regions of 24 amino acids each, which are characterized, according to conformation prediction analyses, by marked alpha-helicity. The sequence shows a single potential N-glycosylation site, which is assigned to the vesicle interior, and a carboxy-terminal tail of 89 amino acids which contains glycine-rich tetrapeptide repeats, the epitope of monoclonal antibody SY38, and a number of collagenase-sensitive sites accessible on the surface of the intact vesicles. These features suggest that the polypeptide spans the vesicle membrane four times, with both N and C termini located on the outer, i.e. cytoplasmic, surface of the vesicles.     

The molecular weight of the extracellular haemoglobin of Lumbricus terrestris determined by electron microscopy

1. The mol. wt of the extracellular haemoglobin of the oligochaete Lumbricus terrestris was determined by counting in negatively stained electron micrographs. 2. The value obtained using apoferritin as a mol. wt standard is (3.8 +/- 0.3) x 10(6), in agreement with recent determinations employing different physical methods. 3. We conclude that all annelid extracellular haemoglobins and chlorocruorins which have the same dimensions as Lumbricus haemoglobin probably have the same mol. wt.     

Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities

Hermetical microwave was used to degrade Porphyridium cruentum polysaccharides from 2918 to 256.2, 60.66 and 6.55 kDa. The antioxidant properties of different molecular weight polysaccharides were evaluated by determining the scavenging ability of free radicals, inhibitory effects on lipid peroxidation in liver homogenates and hemolysis of mouse erythrocytes. Analysis of physicochemical properties confirmed that microwave degradation might not markedly change the chemical components of the polysaccharides. High-molecular-weight polysaccharides from P. cruentum had no obvious antioxidant activity, but low-molecular-weight fragments after degradation exerted an inhibitory effect on oxidative damage. The 6.55-kDa fragment had stronger antioxidant activity than the 60.66 and 256-kDa fragments.