Binding of hyaluronic acid to mammalian fibrinogens

We have postulated that the interaction of hyaluronic acid (HA), an extracellular matrix glycosaminoglycan, with fibrin is important during the early stages of wound healing and inflammation (J. Theor. Biol. 119:219; 1986), and have demonstrated the specific binding of 125I-labeled HA to human fibrinogen (J. Biol. Chem. 261:12 586; 1986). To determine whether HA binding is limited to human fibrinogen, we tested the ability of fibrinogens from various mammalian species to bind 125I-HA using a dot-blot assay. Increasing amounts of fibrinogen were adsorbed to nitrocellulose, and incubated with 125I-HA in the presence or absence of a 100-fold excess of nonradiolabeled HA to assess specific binding. In three independent experiments, the amount of 125I-HA bound/mg fibrinogen was determined from the slope derived by linear regression analysis of specifically bound 125I-HA versus protein concentration. A Student's t-test was performed to determine whether the slopes were statistically greater than zero. HA binding was considered statistically significant when P less than 0.05 was obtained by this analysis. Rabbit and dog fibrinogens significantly bound HA in all three trials. Baboon fibrinogen demonstrated significant HA binding in two of three trials. Pig, sheep and goat fibrinogens bound HA significantly in only one of three trials, whereas horse, rat and cow fibrinogens did not bind HA significantly at all. We conclude that fibrinogen from mammalian species other than human can specifically bind HA. The ability of fibrinogen to bind HA appears to correlate with an evolutionary divergence that separated human, baboon, dog, rabbit and rat from cow, pig, horse, goat and sheep.     

Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells

The production and characterization of an antibody to rat liver CTP:phosphocholine cytidylyltransferase is described. This antibody quantitatively precipitated cytidylyltransferase from both rat liver and HeLa cell cytosol. Following affinity purification, the antibody was used to demonstrate, for the first time, the phosphorylation of cytidylyltransferase in vivo. Following the immunoprecipitation of cytidylyltransferase from HeLa cells, acid hydrolysis, and thin layer electrophoresis of the amino acids, only [32P]phosphoserine was detected. The phosphorylation state of cytidylyltransferase in HeLa cells was examined following treatment with phorbol ester for 1 h. In agreement with previous studies, the incorporation of [3H]choline into phosphatidylcholine via the CDP-choline pathway was stimulated 5-fold in cultures of HeLa cells following treatment with phorbol ester for 1 h. However, no appreciable translocation of cytidylyltransferase was detected, despite the utilization of two different methods of cell lysis. Furthermore, the inclusion of phosphatase inhibitors and chelators of divalent cations in the homogenization buffers had no effect on the observed distribution or activity of the enzyme. Immunoprecipitated cytidylyltransferase was phosphorylated to the same extent, and on serine residues only, in both control and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated cells. Measurement of the pool sizes of the aqueous intermediates of the CDP-choline pathway, following TPA treatment, revealed a modest decrease in the phosphocholine pool only, consistent with an activation of cytidylyltransferase.     

Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed

Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.     

Chemistry of the polysaccharides of the brown seaweed dictyopteris plagiogramma

Laminaran, fucose-containing polysaccharides (‘fucans’) and alginic acid were isolated from Dictyopteris plagiogramma.The laminaran comprised G- and M-chains (ratio 3: 1). The ‘fucans’ were present in four extracts of a four-step sequential extraction procedure and all contained slightly differing proportions of fucose, xylose, galactose, mannose, glucuronic acid residues and half-ester sulphate. Non-reducing chain ends as well as the positions of glycosidic linkages to fucose, xylose and glucuronic acid are the same as previously reported for other ‘fucans’. Galactose and mannose occur mainly as trisubstituted residues with substitution at 0-1, 0-3, 0-4 and at 0-1, 0-3, 0-6, respectively.     

Properties of the Photosynthetic System and DNA of Cyanophora paradoxa Cyanelles

The cyanelle from the photosynthetic biflagellate protist Cyanophora paradoxa has been studied in terms of its photosynthetic properties. Structurally, the cyanelle resembles unicellular cyanobacteria. The cyanelle is readily released from the host cell by means of the French press. The isolated cyanelle shows typical photosystem I and photosystem II activities as well as phenazine methosulfate-mediated photophosphorylation. The kinetic parameters K_m and V_max were determined for CO₂ fixation in the cyanelle and cells of C. paradoxa and compared to a cyanobacterium. The determined values were not much different, although the cyanobacterium had a significantly greater rate of CO₂ fixation, and the cyanelle was least active in this regard. Photosystem I chlorophyll-protein complex is readily isolated from the thylakoid membrane. In all these respects, the photosynthetic apparatus of the cyanelle resembles that of cyanobacteria. No nitrogen fixation activity was observed. Attempts to regenerate the isolated cyanelle were not successful, but in some cases, an unidentified cyanobacterium grew up in standing cultures of C. paradoxa cyanelles. Buoyant density data indicate that the strain of C. paradoxa we have investigated differs from that employed by others, since our strain shows a value of 1.716 grams per cubic centimeter and others report values of 1.695 and 1.691.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

Evidence that the hepatic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature

Rat hepatocytes in the continuous presence of [³H]asialo-orosomucoid quickly establish a steady state number of free and occupied surface receptors and rate of endocytosis. These values do not change even though many times more glycoprotein is internalized than there are surface receptors per cell. However, when cells endocytose only one round of surface bound [³H]asialo-orosomucoid at 37°C the internalization of glycoprotein is about 5 times faster than the increase of functional receptors on the cell surface. At 18°C new surface receptors appear at only 6% of the rate of internalization of pre-bound asialoglycoprotein. The results suggest that reutilization of asialoglycoprotein receptors is preferentially inhibited at low temperature and that receptor-ligand complexes enter the cell.