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Query: UMLS:C0155339 (Brown)
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Thylakoid membranes from several brown algae have been fragmented with the non-ionic detergent, Triton X-100. Three intrinsic chlorophyll-protein complexes with different pigment compositions have been isolated by sucrose density gradient centrifugation. Brown algae contain the photosystem 1 reaction-centre complex, a P700-chlorophyll a-protein which has similar spectroscopic and chemical properties to those of higher plants. This complex represents about 10--20% of the total chlorophyll in all species; the Acrocarpia paniculata complex has a chlorophyll/P700 ratio of 38. Two main light-harvesting complexes have also been isolated, which have properties unique to brown algae. The heavier of these, an orange fraction, is a fucoxanthin-chlorophyll a/c-protein; this complex contains most of the fucoxanthin and has only chlorophyll c2. The other, a green fraction, is a chlorophyll a/c-protein enriched in violaxanthin. Neither of these complexes possesses detectable photosystem 1 or photosystem 2 activities. Both of these complexes efficiently transfer light energy to chlorophyll a, indicating that the molecular arrangement of their pigments is similar to that in vivo. Differential extraction of thylakoid membranes indicates that the P700-chlorophyll a-protein is the complex most firmly embedded in the membrane, but the fucoxanthin-chlorophyll a/c-protein is the least firmly bound. We suggest that the fucoxanthin complex is the most variable component of the photosynthetic unit of brown algal chloroplasts.
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PMID:Chlorophyll-protein complexes of brown algae: P700 reaction centre and light-harvesting complexes. 25 38

Three entirely different tumor types were investigated biochemically for the presence and characteristics of endogenous carbohydrate-binding proteins in an inbred Brown Norway rat, an outbred Sprague-Dawley rat, and an outbred Han:NMRI mouse. The patterns under investigation included specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively, and specificities for heparin, analyzed by affinity chromatography on resins with immobilized sugars or glycoproteins and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to dependence of the binding activity on the presence of Ca2+ and dependence on extraction conditions. Rhabdomyosarcoma revealed only Ca2+-independent activities, i.e., activities with specificity for beta-galactosides at a molecular weight of 12,000, with specificity for alpha-galactosides at molecular weights of 29,000, 43,000, and 45,000, with specificity for heparin at molecular weights of 13,000 and 16,000, and with specificities for mannose and fucose at molecular weights ranging from 62,000 to 70,000. For the spontaneous mammary adenocarcinoma the pattern was entirely different and more diverse, including species with the Ca2+ requirement. Extracts with the use of 0.2 M NaCl (salt) and 2% Triton X-100 (detergent) from teratoma contained at least nine different carbohydrate-binding proteins. The only similarities between the pattern of endogenous carbohydrate-binding proteins from teratoma and from mammary adenocarcinoma were beta-galactoside-binding proteins, one with a Ca2+ requirement and one without a Ca2+ requirement, and the heparin-binding proteins. These heparin-binding proteins were the only types of carbohydrate-binding proteins common to all three tumor types. The analysis indicates that certain bands represented newly identified proteins capable of binding to galactose-, mannose- or fucose-containing glycoconjugates, respectively. When assayed with rabbit erythrocytes, the different fractions showed agglutination activity. They can thus be termed "endogenous lectins." The use of endogenous lectin patterns as potential diagnostic markers in addition to the corresponding changes in the glycoconjugate composition is proposed.
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PMID:Biochemical characterization of endogenous carbohydrate-binding proteins from spontaneous murine rhabdomyosarcoma, mammary adenocarcinoma, and ovarian teratoma. 659 44

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of Fc gamma RII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked Fc gamma RIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of Fc gamma RII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via Fc gamma RIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of Fc gamma RIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of Fc gamma RIIIB with Fc gamma RII overcomes the pertussis toxin inhibition of H2O2 production in response to direct ligation of Fc gamma RII. These data support the hypothesis that activation of Hck upon Fc gamma RIIIB ligation has a role in generation of the synergistic respiratory burst.
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PMID:Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst. 776 58

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-glycoside by the action of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. This enzyme is a soluble cytochrome b5-dependent monooxygenase and has been purified to apparent homogeneity from pig submandibular glands by precipitation with N-cetyl-N,N,N-trimethylammonium bromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose, Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12. This procedure resulted in an 8960-fold purification of the hydroxylase with a recovery of 0.8%. The molecular mass of this protein was shown to be 65 kDa on SDS-PAGE and approximately 60 kDa as determined by gel filtration on Superose S.12, which suggests that the enzyme is a monomer. The purified CMP-Neu5Ac hydroxylase is activated by FeSO4 and inhibited by iron-binding reagents such as o-phenanthroline, KCN, Tiron and ferrozine. An apparent Km of 11 microM was determined for the substrate CMP-Neu5Ac using purified hydroxylase in the presence of Triton X-100-solubilized microsomes. In a reconstituted system consisting of purified hydroxylase, cytochrome b5, cytochrome b5 reductase and catalase, an apparent Km of 3 microM was measured. The apparent Km for cytochrome b5 in this system was 0.24 microM. Immunization of a rabbit with enriched and purified hydroxylase led to an antiserum that inhibited CMP-Neu5Ac hydroxylase activity and reacted with the purified 65 kDa protein on a Western blot after SDS-PAGE. Antibodies specific for this 65 kDa protein were isolated and showed a strong reaction with the purified CMP-Neu5Ac hydroxylase from mouse liver after immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands. 788 Nov 82

Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187-199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low-salt and high-salt detergent-free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X-100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low-salt, high-salt and detergent) resolved almost exclusively as a single peak between 6.9-7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic subunits and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X-100, Brij 96) in the gradient was a component of the detergent-soluble extract, indicating the existence of a minor amphiphilic form. In low-salt-soluble and high-salt-soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate-labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66-70 kDa with the active site being located in a 30-kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this 'non-cholinergenic' nematode secretion.
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PMID:The molecular forms of acetylcholinesterase from Necator americanus (Nematoda), a hookworm parasite of the human intestine. 830 98

In simple epithelial cells, the delivery of apical and basolateral proteins to the cell surface is mediated by sorting in the trans-Golgi network and transport via separate vesicular carriers. In order to identify the molecular machinery involved in protein sorting, we have recently studied a detergent-insoluble complex in Madin-Darby canine kidney (MDCK) cells, following CHAPS extraction of exocytic carrier vesicles, specifically including the apical marker protein influenza hemagglutinin (HA). Previously, a Triton X-100 insoluble membrane residue that was enriched in glycosylphosphatidylinositol-anchored (GPI) proteins and glycolipids was characterized and implicated in transport to the apical cell surface [Brown, D., & Rose, J. (1991) Cell 68, 533-544]. In this report, the protein compositions of the CHAPS and Triton complexes have been compared by two-dimensional gel analysis. Only a few major membrane proteins are found in the complexes. The protein compositions are qualitatively similar, but differ quantitatively in the individual components. The CHAPS complex is depleted of GPI-linked proteins and retains a minor fraction of lipids similar in composition to that of the Triton X-100 insoluble complex. We propose that in vivo the complexes form part of a sorting platform that mediates protein segregation and delivery to the apical cell surface.
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PMID:Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. 851 82

We have investigated the effect of UVC irradiation on "TGF alpha ase" activity using both intact HeLa cells and isolated membrane fragments with an assay based on the previously described nonapeptide substrate method [Brown et al. (1992): J Cell Biochem 48:411-423]. This method allows recognition of cleavage at the scissile bond cognate with that of the TGF alpha N-terminal cleavage site from its membrane-bound precursor. The level of ectoendopeptidase (including "TGF alpha ase") activity observed on intact cells was lower than that of ectoaminopeptidases. Addition of foetal bovine serum (FBS) enhanced aminopeptidase and dipeptidyl peptidase activity but inhibited "TGF alpha ase" activity, while phorbol 12-myristate 13-acetate (PMA) had no significant effect on the ectopeptidases monitored, expect for "TGF alpha ase," which was also inhibited, in contradistinction to their effects in other cell systems. Sublethal UVC irradiation (10 Jm-2) of the cultures resulted in activation of the ectoaminopeptidase and ectoendopeptidases which was maximal 16 and 20-24 h after irradiation, respectively. The addition of FBS to these irradiated cells appeared to reduce the increase in endopeptidase products, due in part to increased aminopeptidase activity but also to the direct inhibitory effect of FBS on the "TGF alpha ase." The activation of these proteases by UVC, even at high fluences (500 Jm-2), was not observed within the first 30 min after the cells were irradiated. Purified plasma membrane fragments were prepared from suspension cultures of HeLa cells and displayed high levels of "TGF alpha ase" activity. The rate of "TGF alpha ase" activity using 140 nM peptide substrate (P9) was 5.6 pmol/min/mg membrane protein, which was elevated to 13.7 pmol/min/mg membrane protein, 20 h after the cells had been irradiated with 10 Jm-2 UVC. Inhibition studies indicate that the plasma membrane "TGF alpha ase is a metalloenzyme as it was inhibited by EDTA, EGTA, and 1,10-phenanthroline but not by elastase or serine protease inhibitors. "TGF alpha ase" activity on intact cells was shown to be inhibited by 1,10-phenanthroline, which further supports this suggestion. Treatment of the membranes with Triton X-100 resulted in a loss of "TGF alpha ase" activity, raising the possibility that this enzyme may require a cofactor to be fully functional. We show that in purified membrane preparations of HeLa cells there is evidence for the presence of a "TGF alpha ase" which can be activated by UV irradiation, which differs from the putative "TGF alpha ase" described in various other cell lines, and which does not seem dependent on protein kinase C (PKC) activity.
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PMID:UVC activation of the HeLa cell membrane "TGF alpha ase," a metalloenzyme. 905 93

Treatment of cultured neurons with non-ionic detergents under certain conditions causes the axonal microtubules to splay apart from each other, allowing individual microtubules to be visualized by immunofluorescence microscopy [Brown et al., 1993, J. Cell Sci. 104: 339-352]. I have investigated whether axonal neurofilaments separate from each other under similar conditions. Cultures of dissociated dorsal root ganglion (DRG) neurons from fetal rats were treated with non-ionic detergent and fixed with formaldehyde. Neurofilaments were visualized by immunofluorescence microscopy using a polyclonal antiserum specific for NF-L. Treatment of the neurons with Triton X-100 or saponin caused filamentous structures to splay apart from each other along the entire length of the axon. Quantitative analysis of fluorescence intensity along the filamentous structures indicated that many of them represent single neurofilaments and that single and bundled neurofilaments can be distinguished based on their fluorescence intensity. The extent of this splaying phenomenon was dependent on time and detergent concentration. Temporal analysis indicated that short portions of single neurofilaments initially loop out from the axonal bundle and then subsequently splay apart further along their length and adhere to the polylysine/laminin coated substrate. The maximum observed length for a single axonal neurofilament was 183 microm in neurons after only 1 day in culture, which indicates that neurofilaments can attain remarkable lengths in these young cultured neurons. The splayed axonal cytoskeleton preparation described here allows individual axonal neurofilaments to be visualized by immunofluorescence microscopy, which is not possible in conventional preparations due to the dense packing of these polymers in axons.
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PMID:Visualization of single neurofilaments by immunofluorescence microscopy of splayed axonal cytoskeletons. 933 Dec 18

Domains rich in sphingolipids and cholesterol, or rafts, may organize signal transduction complexes at the plasma membrane. Raft lipids are believed to exist in a state similar to the liquid-ordered phase. It has been proposed that proteins with a high affinity for an ordered lipid environment will preferentially partition into rafts (Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910-3917). We investigated the possibility that lipid-lipid interactions between lipid-modified proteins and raft lipids mediate targeting of proteins to these domains. G protein monomers or trimers were reconstituted in liposomes, engineered to mimic raft domains. Assay for partitioning of G proteins into rafts was based on Triton X-100 insolubility. Myristoylation and palmitoylation of Galpha(i) were necessary and sufficient for association with liposomes and partitioning into rafts. Strikingly, the amount of fatty-acylated Galpha(i) in rafts was significantly reduced when myristoylated Galpha(i) was thioacylated with cis-unsaturated fatty acids instead of saturated fatty acids such as palmitate. Prenylated betagamma subunits were excluded from rafts, whether reconstituted alone or with fatty-acylated alpha subunits. These results suggest that the structural difference between lipids that modify proteins is one basis for the selectivity of protein targeting to rafts.
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PMID:Lipid-dependent targeting of G proteins into rafts. 1063 25

Trichomonas vaginalis has been reported to possess alternative 2-keto acid oxidoreductases (KORs). These enzymes preferentially used indolepyruvate in a reaction that resembled that of pyruvate:ferredoxin oxidoreductase (PFO). However, the KORs did not reduce ferredoxin and remained active in metronidazole-resistant trichomonads lacking PFO. Therefore, it was proposed that the KORs may help trichomonads to survive in the presence of metronidazole. The KORs were identified using activity staining on native gels (Brown DM, Upcroft JA, Dodd HN, et al. Alternative 2-keto acid oxidoreductase activities in T. vaginalis. Mol Biochem Parasitol 1999;98:203-14). In the current study, we showed that the apparent KOR activity was caused by the non-enzymatic reduction of the indicator dye, nitroblue tetrazolium, by indolepyruvate, which is facilitated by Triton X-100 used to prepare the membrane fractions. We could not confirm the presence of KORs in metronidazole-resistant T. vaginalis. The low level indolepyruvate-dependent activity that is present in T. vaginalis strains sensitive to metronidazole is catalyzed by PFO, which was verified using the pure enzyme. Therefore, our results suggest that alternative 2-keto acid oxidoreductases do not exist in T. vaginalis.
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PMID:Alternative 2-keto acid oxidoreductases in Trichomonas vaginalis: artifact of histochemical staining. 2196 39


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