Sulfated polysaccharides from the red seaweed Georgiella confluens.

The water-soluble polysaccharides from Georgiella confluens, collected in Antarctica, were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The initially extracted polysaccharide and the major fraction isolated, soluble in 0.5 M NaCl, were studied. These are sulfated xylogalactans naturally rich in methylated sugar residues, comprising of 3,6-anhydro-2-O-methyl-L-galactose, 2-O-methyl-L-galactose and 6-O-methyl-D-galactose. Structural analysis was carried out by methylation, ethylation, desulfation-ethylation, desulfation-methylation, Smith degradation, 13C NMR spectroscopy and determination of the absolute configuration of monosaccharides by gas chromatography of diastereomeric derivatives produced by reductive amination. The results indicated the presence of an agaran backbone with an unusual substitution pattern: sulfation mainly at the 3-position of the alpha-L-galactose units and the presence of xylose side chains at the 4-position of the beta-D-galactose residues.     

Seasonal photosynthetic performance of the endemic antarctic red algaPalmaria decipiens (Reinsch) Ricker

 A male gametophyte of the endemic Antarctic red macroalga Palmaria decipiens (Reinsch) Ricker was cultivated under fluctuating daylengths, simulating the seasonal changes at the site of collection (King George Island, Antarctica). The plant was maintained at 0±1°C, an irradiance of 25 μmol m^-2 s^-1 and under growth-saturating nutrient conditions. Samples were taken at intervals of 3–6 weeks to measure growth, photosynthesis, dark respiration and pigment content. The growth optimum in spring coincided with a higher photosynthetic activity. Whereas dark respiration was constantly low over the year, there was a rapid increase in maximum photosynthetic rate (P_max) in conditions corresponding to September and October. This was correlated with a change in the initial slope (α) of the photosynthesis versus irradiance (P vs I) curve. Higher activity in photosynthesis mainly resulted from higher Chl a and phycobilin concentrations during Antarctic spring, an indication of an increase in absorption cross-section areas of photosynthetic reaction centres. These changes in physiology are discussed in relation to the seasonal growth “strategy” of the species, which is controlled by seasonal variation in daylength. Received: 27 February 1995/Accepted 3 October 1995     

Primate (Macaca fascicularis) transferrin: isolation and partial characterization

The serum transferrin from the primate, Macaca fascicularis is isolated by a purification protocol consisting of ammonium sulphate precipitation and column chromatography. The hexose (galactose + mannose) content of Macaca transferrin is 4.7 mole per mole of protein. Quantitative determination of the sialic acid content shows that there are two sialic acid residues per molecule of Macaca transferrin. This conclusion is supported by the neuraminidase treatment of Macaca transferrin, in which there is a 2-step decrease in electrophoretic mobility. Monoferric Macaca transferrins with Fe3+ selectively labelled at the C- and N-terminal sites (TfFec and FeNTf) are prepared at pH 5.5 and 8.5 using ferric dinitrilotriacetate [Fe(NTA)2] chelate and ferrous ammonium sulphate, respectively.     

Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit M(r) 22,000 having an optimum catalytic activity at pH 6.5-7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for K(m)(GST) and K(m)(CDNB) of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 degrees C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins.     

Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.     

Carbohydrates of the antarctic brown seaweed Ascoseira mirabilis

Mannitol, sucrose and four monosaccharides were obtained from an ethanolic extract of Ascoseira mirabilis. Sequential extraction with aqueous calcium chloride, dilute acid and dilute alkali gave mixtures of laminaran, ‘fucan’ and alginic acid. Laminarans fractionated from the extracts contained different proportions of uniformly (1 → 3) and (1 → 6) linked chains of β-D-glucose residues. The ‘fucan’ contained varying proportions of fucose, galactose and glucuronic acid, small amounts of xylose, mannose, glucose, half ester sulphate and protein. Extraction of the weed under mild alkaline conditions gave a yield of 13.4% of low molecular weight calcium alginate with a mannuronate to guluronate ratio of 30:70 and only a small proportion of sequences of alternating residues. Selective extraction and fractionation gave alginate fractions rich (> 80%) in mannuronate or guluronate.     

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.     

DNA isolation protocol for red seaweed (rhodophyta)

We report a DNA isolation protocol for red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 μg of high molecular weight DNA from 10 mg of dried material, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

The isolation and partial characterization of chymotrypsinogen from the pancreas of the ostrich (Struthio camelus)

1. Cationic chymotrypsinogen from the pancreas of the ostrich was purified to homogeneity by sulfuric acid extraction of pancrei, (NH4)2SO4 fractionation and SP-Sephadex C-50 and Sephadex G-100 chromatography. 2. The final preparation was homogeneous when subjected to SDS-PAGE, isoelectric focusing and sedimentation equilibrium centrifugation. The Mmin value obtained from amino acid analysis was 25,572 Da. A mean sedimentation coefficient of 2.575 S was obtained by sedimentation velocity centrifugation. 3. N-terminal analysis by dansylation showed an Ala residue which is the N-terminal of a neochymotrypsinogen. 4. The effects of pH, temperature and inhibitors (LBTI, PMSF, TPCK and DFP) on the chymotryptic activity were examined. A Km-value for ATEE as substrate was found to be 0.57 mM.     

The isolation and some properties of a lectin (Haemagglutinin) from Cucurbita phloem exudate

The occurrence of high haemagglutinating (lectin) activity in phloem exudate from three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita maxima Duch. has been isolated by cation exchange chromatography on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 g/ml. No sugar, including those transported in the phloem of these species, interacted with agglutination. The lectin could not be extracted from cucurbit seed, but appeared in 5-day old seedlings. The possible role of a lectin in the sieve element is discussed.     

Cuticle of Caenorhabditis elegans: its isolation and partial characterization

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross-linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.     

Messenger RNA of human immune interferon: isolation and partial characterization

Suspension cultures of total and lymphocyte-enriched peripheral white blood cells were used as a source for the preparation of mRNA for human γ-interferon. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose and further purified on a preparative sucrose gradient. The RNA preparations were translated by oocytes into (a) protein(s) that showed biological activity in the interferon-assay. On sucrose gradient centrifugation the translatable fractions of the RNA migrated with a sedimentation coefficient of approximately 15 S, a value compatible with the molecular weight of human γ-interferon. The translation product was further characterized by serological cross reactivity with purified γ-interferon in neutralization reactions.     

The plasma membrane of Streptococcus cremoris: isolation and partial characterization

Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase.     

Ascaris suum: partial isolation and characterization of hypodermis from the adult female

A method was developed to remove the muscle from body wall strips of adult female Ascaris suum resulting in a hypodermis cuticle preparation. Optimum treatment for obtaining the hypodermis cuticle was a 15 min incubation with trypsin (2.0 mg/ml) at room temperature, followed by mechanical removal of the muscle. The hypodermis cuticle prepared in this manner incorporated radiolabeled amino acids into cuticular and hypodermal proteins; incorporation was inhibited by protein synthesis inhibitors. Characterization of the hypodermal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the hypodermis apparently contains proteins that differ from those of the cuticle and that the hypodermis of adult A. suum appears to lack cuticle protein precursors. This result will now allow detailed biochemical and physiological investigations of the hypodermis, a tissue which is critical for cuticle synthesis.     

Collagen polymorphism: isolation and partial characterization of alpha 1(I)-trimer molecules in normal human skin.

Human skin has previously been shown to contain at least two genetically distinct types of collagen, type I and III. Here the presence of an additional form of collagen, α1(I)-trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 m NaCl at pH 7.4. The α-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 m NaCl precipitates contained predominantly type I collagen with a chain composition of [α1(I)]₂α2. In the 1.5 m precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 m NaCl fraction consisted almost exclusively of α-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with α1(I). The amino acid composition of these chains was also similar to that of α1(I), except that hydroxylysine was increased and lysine was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the α-chains in α1(I)-trimer are the same gene products as α1 in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in α1(I)-trimer. Estimation of the quantitative amounts of α1(I)-trimer indicated that this collagen accounts for less than 5% of the total collagen in adult human skin. It is speculated, however, that α1(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.     

Experimental allergic orchitis: the isolation and partial characterization of an aspermatogenic polypeptide (AP3) with an apparent sequential disease-inducing determinant(s)

An aspermatogenic polypeptide (AP3) capable of inducing experimental allergic orchitis (EAO) in the guinea pig (GP) was purified from GP testes by sequential delipidation, acid extraction, pH precipitation, ammonium sulfate fractionation, trichloroacetic acid precipitation, gel filtration on Sephadex G-75, preparative isoelectric focusing from pH 3-10 followed by isoelectric focusing from pH 7-10, gel filtration on Sephadex G-75 Superfine under reducing conditions, and reduced acid urea gel electrophoresis. Approximately 250 micrograms (BSA equivalents) of AP3 were obtained from 500 g wet weight of GP testes. On 15% reduced acid urea polyacrylamide gels, AP3 appeared as a single band with an Rf of 0.19. SDS-PAGE showed a single band with a mobility corresponding to a m.w. of 12,500 +/- 1500. The isoelectric point, determined during purification, was 9.90 +/- 0.50. Amino acid analysis of AP3 indicates it is a protein. Gas liquid chromatographic analysis failed to reveal the presence of either hexose or hexosamine, indicating that AP3 is probably not a glycopeptide. Two to 5.0 micrograms (BSA equivalents) of AP3 are capable of inducing severe EAO in 100% of GP tested; 1 to 2.0 micrograms (BSA equivalents) induced EAO in 60% of GP tested. Because AP3 appears to be nonglycosylated and the aspermatogenic activity of AP3 is highly resistant to various denaturing conditions including reduction and alkylation, the primary sequence of the polypeptide rather than higher ordered structure may be more important in defining the determinant(s) responsible for its aspermatogenic activity.