Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Compartmentalisation of nitrogenase in a non-heterocystous cyanobacterium: Trichodesmium contortum

Abstract In Trichodesmium contortum , nitrogenase was detected in only a limited number (about 10%) of microscopically distinguishable, consecutively arranged cells in central regions of the trichomes. Cells with nitrogenase also contained the photosystem II associated pigment phycoerythrin. These cells were not distinguishable from other cells on a structural basis, but were clearly visible at low magnification microscopy as all in the zone were more compact and shorter than those on either side. The compartmentalisation of nitrogenase into a chain of cells and in a possibly photosynthetic environment represents a previously undescribed phenomenon. The nitrogenase containing cells apparently perform the O₂ protective function of heterocysts yet are different in several aspects.     

Phylogenetic analyses of primate size evolution: the consequences of sexual selection

We have analysed the relationship between primate mating system, size and size dimorphism by utilizing several phylogenetically based methods. An independent contrast analysis of male and female size (log weight) showed that these are tightly correlated and that size dimorphism is not a simple allometric function of size. We found no relationship between mating system and sexual dimorphism in strepsirhines but a strong relationship in haplorhines. By matched-pairs analysis, where sister groups were matched according to whether the mating system predicted higher or lower intrasexual selection for male size, haplorhine species in more polygynous clades (with a predicted higher sexual selection) were significantly more dimorphic, had larger males, and also, but to a lesser degree, larger females. Both independent contrast and matched-pairs analyses are non-directional and correlational. By using a directional test we investigated how a transition in mating system affects size and dimorphism. Here, each observation is the sum of changes in dimorphism or size in a clade that is defined by a common origin of a mating system. Generally, dimorphism, as well as male and female size, increased after an expected increase in sexual selection, and decreased after an expected decrease in sexual selection. The pattern was, however, not significant for all of the alternative character reconstructions. In clades with an expected increase in sexual selection, male size increased more than female size. This pattern was significant for all character reconstructions. The directional investigation indicates that the magnitude of change in haplorhine dimorphism is larger after an increase in sexual selection than after a decrease, and, for some reconstructions, that the magnitude of size increase is larger than the magnitude of size decrease for both sexes. Possible reasons for these patterns are discussed, as well as their implications as being one possible mechanism behind Cope's rule, i.e. general size increase in many phylogenetic lineages.     

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Norepinephrine Metabolism in Humans Studied by Deuterium Labelling: Turnover of 4–Hydroxy-3–methoxyphenylglycol

Abstract: D, L(±)-4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study turnover of plasma free HMPG following an intravenous injection. Ten healthy men were given a pulse dose of either 4.3 μmol or 2.2 μmol of labelled HMPG ([²H₃]HMPG piperazine salt). Plasma and urine levels of both endogenous and labelled HMPG were subsequently followed by gas chromatography-mass spectrometry with selected ion detection. Kinetic calculations based upon a single-compartment model were consistent with a monoexponential elimination of plasma free HMPG. The half-life of HMPG was 0.46 and 0.78 h (mean values in the two dose groups). The HMPG production rate was 2.01 and 2.35 μmol/hour, and the urinary excretion rate of HMPG (free and conjugated) was 0.48 and 0.47 μmol/h. The endogenous plasma level of free HMPG was 25 and 33 nmol/L. The results show that HMPG turns over rapidly and that HMPG is further metabolized extensively. About one-fourth of the HMPG produced is excreted in urine as free and conjugated HMPG.     

The copolymeric structure of pig skin dermatan sulphate. Isolation and characterization of l-idurono-sulphate-containing oligosaccharides from copolymeric chains

Dermatan sulphate was degraded by testicular hyaluronidase and an oversulphated fraction was isolated by ion-exchange chromatography. This preparation, which contained fairly long segments derived from the non-reducing terminal portion of the molecule, was subjected to periodate oxidation under acidic conditions. The oxidized iduronic acid residues were cleaved by reduction-hydrolysis (Smith-degradation) (Fransson & Carlstedt, 1974) or by alkaline elimination. The oligosaccharides so obtained contained both GlcUA (glucuronic acid) and IdUA-SO(4) (sulphated iduronic acid) residues. Copolymeric oligosaccharides obtained after alkaline elimination were cleaved by chondroitinase-AC into disaccharide and higher oligosaccharides. Since the corresponding oligosaccharides obtained by Smith-degradation were unaffected by this enzyme, it was concluded that the carbohydrate sequences were GalNAc-(IdUA-GalNAc)(n)-GlcUA-GalNAc. The iduronic acid-containing sequences were resistant to digestion with chondroitinase-ABC. It was demonstrated that the presence of unsulphated N-acetylgalactosamine residues in these sequences could be responsible for the observed effect. This information was obtained in an indirect way. Chemically desulphated dermatan sulphate was found to be a poor substrate for the chondroitinase-ABC enzyme. Moreover, digestion with chondroitinase-ABC of chondroitinase-AC-degraded dermatan sulphate released periodate-resistant iduronic acid-containing oligosaccharides. It is concluded that copolymeric sequences of the following structure are present in pig skin dermatan sulphate: [Formula: see text] N-acetylgalactosamine moieties surrounding IdUA-SO(4) residues are unsulphated to a large extent.     

The ultrastructural localization of a cholinesterase in the body muscle of plaice (Pleuronectes platessa)

Summary By means of a histochemical method adapted for electron microscopy a cholinesterase in body muscle cells of plaice (Pleuronectes platessa) has been localized to the sarcolemma. The cholinesterase activity disappeared from the sarcolemma after the muscle tissue had been incubated with a bacterial enzyme, which had earlier been shown, by biochemical methods, to be able to liberate this cholinesterase activity from plaice muscle.The provision of live plaices from Kristineberg Zoological Station, Fiskebäckskil, Sweden, is gratefully acknowledged. We are greatly indebted to Dr. Åke Bovallius, FOA, who provided the starting material for the bacterial enzyme. We would like to express our sincere thanks to Prof. Lennart Nicander for valuable discussions and for placing the resources of the Department of Anatomy and Histology, Royal Veterinary College, to our disposal.