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Effects of surfactant and boron doping on the BWF feature in the Raman spectrum of single-wall carbon nanotube aqueous dispersions.

We examine the Breit-Wigner-Fano (BWF) line shape in the Raman spectra of carbon single-wall nanotubes (SWNTs) dispersed in aqueous suspensions. Bundling and electronic effects are studied by comparing undoped SWNTs (C-SWNTs) to boron-doped nanotubes (B-SWNTs) in a variety of different surfactant solutions. For SWNTs dispersed with nonionic surfactants that are less effective in debundling than ionic surfactants, the Raman spectra retain a large BWF feature. However, we demonstrate that even for SWNTs dispersed as isolated nanotubes by ionic surfactants the BWF feature may be present and that the intensity of the BWF is highly sensitive to the specific surfactant. In particular, surfactants with electron-donating groups tend to enhance the BWF feature. Also, modification of the SWNT electronic properties by boron doping leads to enhanced surfactant dispersion relative to undoped C-SWNTs and also to modification of the BWF feature. These observations are in agreement with reports demonstrating an enhancement of the BWF by bundling but also agree with reports that suggest electron donation can enhance the BWF feature even for isolated SWNTs. Importantly, these results serve to caution against using the lack or presence of a BWF feature as an independent measure of SWNT aggregation in surfactant dispersions.

Benzenesulfonates↗

Spectroscopy of single- and double-wall carbon nanotubes in different environments.

Individual single-wall carbon nanotubes (SWNTs) and double-wall carbon nanotubes (DWNTs) were suspended in water for optical studies using sodium-cholate and other surfactants. We used time-resolved photoluminescence (PL) spectroscopy to study the influence of tube chirality and diameter as well as of the environment on nonradiative decay in small diameter tubes. The studies provide evidence for PL from small diameter core tubes in DWNTs and for a correlation of nonradiative decay with tube diameter and exciton red shift as induced by interaction with the environment.

Light↗

Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes.

Human cytochromes P450 (P450) 1A2 and P450 3A4 were expressed in Escherichia coli, purified, and used in reconstituted oxidation systems. The optimal system for P450 3A4 included a mixture of phospholipids, sodium cholate, cytochrome b5, GSH, and MgCl2. Relatively high catalytic activities were obtained with such a system for aflatoxin (AF) B1 3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active than P450 1A2 in forming genotoxic AFB1 oxidation products. Analysis of the AFB1 products indicated that P450 3A4 formed AFQ1 and the exo-8,9-epoxide; P450 1A2 formed AFM1, a small amount of AFQ1, and both the exo- and endo-8,9-epoxides. The endo epoxide is essentially nongenotoxic in the umu test, as found previously in bacterial mutagenicity assays [Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 1603-1609]. 7,8-Benzoflavone (alpha-naphthoflavone, alpha NF) stimulated AFB1 (exo) 8,9-epoxidation and inhibited 3 alpha-hydroxylation in human liver microsomes and a reconstituted P450 3A4 system but was a potent inhibitor of all reactions catalyzed by P450 1A2. Plots of AFB1 3 alpha-hydroxylation and 8,9-epoxidation vs AFB1 concentration were sigmoidal in both human liver microsomes and the reconstituted P450 3A4 system. The results are consistent with the view that P450 3A4 is a major human liver P450 enzyme involved in AFB1 activation, although the in vivo situation may be more complex due to the presence of the enzyme in the gastrointestinal tract.

Aflatoxin B1↗

Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase.

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.

Animals↗

Cloning of Aspergillus fumigatus histidine kinase gene fragment and its expression during invasive infection.

It was demonstrated recently that three histidine kinases genes in Candida albicans contributed to virulence, indicating the importance of signaling pathways regulated by histidine kinases. In the present study, using a set of degenerate primers, RT-PCR was performed with cDNA of A. fumigatus as a template. PCR products were cloned and sequenced. After Blast analysis, it was found that one fragment (named as AFHK1), 305 bp, was highly homologous to the two-component histidine kinase tesA gene of Aspergillus nidulans. But AFHKI was not completely identical to the FOS-1 gene of A. fumigatus. The same A. fumigatus strain was used to inoculate the mice for a murine model of invasive pulmonary aspergillosis (IPA). After 5-days post-inoculation, the lungs of infected animals were removed and incubated for 2 h at 37 degrees C in digestion buffer containing collagenase and trypsin. The pulmonary cells were removed by passing the suspension through a sieve. The non-filterable hyphae were treated with deoxygenated sodium cholate. Total RNA of A. fumigatus isolated from the infected tissues or cultured in vitro was extracted. With AFHKI as a probe. a Northern blot was performed. A 3.0 kb (approximate) transcript of mRNA was detected corresponding to the putative histidine kinase gene. It was demonstrated that that gene was expressed at markedly higher levels in vivo than in vitro. The results suggest that this gene may contribute to the survival and virulence of A. fumigatus.

Animals↗

The relationship between rat intestinal permeability and hydrophilic probe size.

PURPOSE: The relationship between rat intestinal permeability (Papp) of a range of hydrophilic probe molecules and probe geometry was examined. METHODS: Molecules studies included mannitol, the polyethylene glycols (PEGs) 400, 900, and 4000, the dextran conjugated dye Texas Red (MW 3000) and the polysaccharide inulin (MW 5500). Molecular surface area, volume and cross-sectional diameter for each probe were determined from computer models. The effect of the bile salt sodium cholate, and bile salt: fatty acid mixed micelles on probe intestinal permeability was also studied. RESULTS: Of the size parameters tested, cross-sectional diameter correlated best with log intestinal permeability. The data was fitted to a relationship of the form Papp = Papp zero exp(-Krca) where rca is the molecular cross sectional radius. Papp zero and K are constants. Estimates of equivalent pore radii (R) were also made; the use of rca giving the most reasonable estimate of R. Absorption of all probes was enhanced by both simple and mixed micellar systems. CONCLUSIONS: For large hydrophilic probes and possibly protein drugs, cross sectional diameter is a more important size parameter than volume based values for evaluating size-related retarded absorption. The relationship established may be used as a tool to assess absorption enhancement potential of excipients.

Animals↗

Acacia-gelatin microencapsulated liposomes: preparation, stability, and release of acetylsalicylic acid.

Liposomes of dipalmitoylphosphatidylcholine (DPPC) containing acetylsalicylic acid (ASA) have been microencapsulated by acacia-gelatin using the complex coacervation technique as a potential oral drug delivery system. The encapsulation efficiency of ASA was unaltered by the microencapsulation process. The stability of the microencapsulated liposomes in sodium cholate solutions at pH 5.6 was much greater than the corresponding liposomes. The optimum composition and conditions for stability and ASA release were 3.0% acacia-gelatin and a 1- to 2-hr formaldehyde hardening time. Approximately 25% ASA was released in the first 6 hr from microencapsulated liposomes at 23 degrees C and the kinetics followed matrix-controlled release (Q varies; is directly proportional to t1/2). At 37 degrees C, this increased to 75% released in 30 min followed by a slow constant release, likely due to lowering of the phase transition temperature of DPPC by the acacia-gelatin to near 37 degrees C. At both temperatures, the release from control liposomes was even more rapid. Hardening times of 4 hr and an acacia-gelatin concentration of 5% resulted in a lower stability of liposomes and a faster release of ASA. It is concluded that under appropriate conditions the microencapsulation of liposomes by acacia-gelatin may increase their potential as an oral drug delivery system.

1,2-Dipalmitoylphosphatidylcholine↗

Phosphatidate phosphatase--a key enzyme in glycerolipid biosynthesis. Studies on the yeast enzyme.

Phosphatidate phosphatase is an important enzyme in glycerolipid biosynthesis, but difficult to purify. A purified preparation of N-ethylmaleimide-sensitive phosphatidate phosphatase from the yeast Saccharomyces cerevisiae was obtained by a five-step protocol, using chromatography on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superdex 200. A protease-deficient yeast strain gave preparations similar to those of the wild-type strain. In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same position as albumin. However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degradation during purification. A 35-kDa and a 40-kDa protein band which coincided with activity were found in all preparations. Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6% sodium cholate in the buffers was essential.

Electrophoresis, Polyacrylamide Gel↗

Purification and characterization of a novel enantioselective hydrolase from Bacillus subtilis.

Screening of the micro-organisms from an in-house microbial culture repository, identified a bacterial strain bearing membrane-bound, inducible ester hydrolase activity. The strain designated as RRL-BB1 has been identified as Bacillus subtilis by 16 S rRNA typing. Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity. The enzyme, designated as BBL, exhibited high enantioselectivity (ee approximately 99%) with acyl derivatives of unsubstituted and substituted 1-(phenyl)ethanols and 1-(6-methoxy-2-naphthyl)ethanols. With acyl derivatives of 2-(6-methoxy-2-naphthyl)propan-1-ol, moderate enantioselectivity was observed. The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e. 2-bromopropanoic acid and 2-hydroxy-4-phenylbutanoic acid. The enzyme was purified to >90% purity from cell-free extract of RRL-BB1 with 26% overall yield. The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile. A specific activity of 450 units/mg protein resulted after at least a 200-fold purification of the crude cell-free extract. The key purification step was the irreversible adsorption of the salt-precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate. The purified enzyme was a 45 kD monomer as shown by SDS/PAGE. The N-terminal amino acid sequence of the purified enzyme was determined as Thr-Lys-Leu-Thr-Val-Gln-Thr-Arg-Asp-Gly-Ala-Leu-Arg-Gly-Thr. The N-terminal sequence did not bear any homology with other known bacterial lipases. BBL is maximally active at 37 degrees C, pH 8.0 and fairly stable up to 40 degrees C, pH 6-10. The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF. Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).

Bacillus subtilis↗

Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.

Animals↗

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

Alkalies↗

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

3',5'-Cyclic-AMP Phosphodiesterases↗

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.

Animals↗

A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.

Binding Sites↗

The insulin- and glucagon-stimulated 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity.

The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].

3',5'-Cyclic-AMP Phosphodiesterases↗

Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

Adenosine↗

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.

Cell Membrane↗

Improved assay method for activity of thyroid peroxidase-catalysed coupling of iodotyrosine residues of thyroglobulin utilizing h.p.l.c. for analysis of iodothyronines.

The coupling of iodotyrosine residues of thyroglobulin (Tg) catalysed by thyroid peroxidase (TPO) has scarcely been studied with respect to the TPO of abnormal human thyroid glands. The present paper proposes a rapid and convenient assay method applicable for determining the coupling activity of a sample of less than 500 mg from each patient's thyroid. The main characteristics of the method are as follows: (i) mitochondrial/microsomal fractions of thyroid glands were treated with sodium cholate plus trypsin, and the supernatants obtained by ultracentrifugation were directly used for the assay of coupling and peroxidase activity of TPO; (ii) the formation of iodotyrosine residues catalysed by TPO was performed by using chemically iodinated Graves'-disease Tg containing 41 iodine atoms per molecule and with a high iodotyrosine and a low iodothyronine content; (iii) newly synthesized iodothyronine residues (thyroxine, 3,5,3'-tri-iodothyronine, and 3,3',5'-tri-iodothyronine) were analysed by h.p.l.c. after hydrolysis of Tg with proteinases and extraction of iodothyronines with ethyl acetate.

Chromatography, High Pressure Liquid↗