Absence of substrate channeling in the glycosome of Trypanosoma brucei

Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966–6973]. The crosslinked enzyme compl...

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Veröffentlicht in:Molecular and biochemical parasitology 1986-04, Vol.19 (1), p.1-10
Hauptverfasser: Aman, Rashid A., Wang, Ching C.
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description Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966–6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of α-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of α-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5–5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form.
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(1985) J. Biol. Chem. 260, 6966–6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of α-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of α-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5–5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. 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Psychology ; Glucose - metabolism ; Glycerophosphates - metabolism ; Glycolysis ; Glycolytic enzymes ; Human protozoal diseases ; Infectious diseases ; Medical sciences ; Microbodies ; Microbodies - enzymology ; Multienzyme complex ; Multienzyme Complexes - metabolism ; NAD - metabolism ; Oxidation-Reduction ; Parasitic diseases ; Protozoa ; Protozoal diseases ; Spectrophotometry ; Subcellular compartmentation ; Tropical medicine ; Trypanosoma brucei ; Trypanosoma brucei brucei ; Trypanosoma brucei brucei - enzymology ; Trypanosoma brucei brucei - ultrastructure ; Trypanosomiasis</subject><ispartof>Molecular and biochemical parasitology, 1986-04, Vol.19 (1), p.1-10</ispartof><rights>1986</rights><rights>1986 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c441t-cc9c1a01f2affe8f668370a8fbdc1575730742f3c7040df7b67e5dda7db8bbb93</citedby><cites>FETCH-LOGICAL-c441t-cc9c1a01f2affe8f668370a8fbdc1575730742f3c7040df7b67e5dda7db8bbb93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0166-6851(86)90059-9$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&amp;idt=8653220$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3012332$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Aman, Rashid A.</creatorcontrib><creatorcontrib>Wang, Ching C.</creatorcontrib><title>Absence of substrate channeling in the glycosome of Trypanosoma brucei</title><title>Molecular and biochemical parasitology</title><addtitle>Mol Biochem Parasitol</addtitle><description>Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966–6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of α-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of α-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5–5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form.</description><subject>2-Deoxyglucose</subject><subject>Adenosine Triphosphate - metabolism</subject><subject>Animals</subject><subject>Biochemistry. Physiology. Immunology. Molecular biology</subject><subject>Biological and medical sciences</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cross-Linking Reagents</subject><subject>Deoxyglucose - metabolism</subject><subject>Dimethyl Suberimidate</subject><subject>enzymes</subject><subject>Fructose-Bisphosphatase - metabolism</subject><subject>Fructosediphosphates - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glucose - metabolism</subject><subject>Glycerophosphates - metabolism</subject><subject>Glycolysis</subject><subject>Glycolytic enzymes</subject><subject>Human protozoal diseases</subject><subject>Infectious diseases</subject><subject>Medical sciences</subject><subject>Microbodies</subject><subject>Microbodies - enzymology</subject><subject>Multienzyme complex</subject><subject>Multienzyme Complexes - metabolism</subject><subject>NAD - metabolism</subject><subject>Oxidation-Reduction</subject><subject>Parasitic diseases</subject><subject>Protozoa</subject><subject>Protozoal diseases</subject><subject>Spectrophotometry</subject><subject>Subcellular compartmentation</subject><subject>Tropical medicine</subject><subject>Trypanosoma brucei</subject><subject>Trypanosoma brucei brucei</subject><subject>Trypanosoma brucei brucei - enzymology</subject><subject>Trypanosoma brucei brucei - ultrastructure</subject><subject>Trypanosomiasis</subject><issn>0166-6851</issn><issn>1872-9428</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U9r2zAYBnAxOtIs6zdYmQ9jtAev-mdJvhRKaNZBYYc1ZyHJr1INx04le5BvP7kJObYHIcT704N4hNAXgn8QTMRNXqIUqiJXSlzXGFd1WX9Ac6IkLWtO1Rman8g5-pTSX5yRFGKGZgwTyhido9WdTdA5KHpfpNGmIZoBCvdsug7a0G2K0BXDMxSbdu_61G9f4VPc70w3HU1h4-ggfEYfvWkTXBz3BVqv7p-WD-Xj75-_lnePpeOcDKVztSMGE0-N96C8EIpJbJS3jSOVrCTDklPPnMQcN15aIaFqGiMbq6y1NVug74fcXexfRkiD3obkoG1NB_2YtBRKcC7fh4RXXIpKZcgP0MU-pQhe72LYmrjXBOupZz2VqKcStRL6tWc95V8e80e7heZ06Vhsnn87zk1ypvXRdC6kE1OiYpTizL4emDe9NpuYyfoPxSTHcKEoE1ncHgTkWv8FiDq5MP1YEyK4QTd9ePul_wHC7qJ5</recordid><startdate>19860401</startdate><enddate>19860401</enddate><creator>Aman, Rashid A.</creator><creator>Wang, Ching C.</creator><general>Elsevier B.V</general><general>Elsevier Science</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>M7N</scope><scope>7X8</scope></search><sort><creationdate>19860401</creationdate><title>Absence of substrate channeling in the glycosome of Trypanosoma brucei</title><author>Aman, Rashid A. ; Wang, Ching C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c441t-cc9c1a01f2affe8f668370a8fbdc1575730742f3c7040df7b67e5dda7db8bbb93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>2-Deoxyglucose</topic><topic>Adenosine Triphosphate - metabolism</topic><topic>Animals</topic><topic>Biochemistry. Physiology. Immunology. Molecular biology</topic><topic>Biological and medical sciences</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cross-Linking Reagents</topic><topic>Deoxyglucose - metabolism</topic><topic>Dimethyl Suberimidate</topic><topic>enzymes</topic><topic>Fructose-Bisphosphatase - metabolism</topic><topic>Fructosediphosphates - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glucose - metabolism</topic><topic>Glycerophosphates - metabolism</topic><topic>Glycolysis</topic><topic>Glycolytic enzymes</topic><topic>Human protozoal diseases</topic><topic>Infectious diseases</topic><topic>Medical sciences</topic><topic>Microbodies</topic><topic>Microbodies - enzymology</topic><topic>Multienzyme complex</topic><topic>Multienzyme Complexes - metabolism</topic><topic>NAD - metabolism</topic><topic>Oxidation-Reduction</topic><topic>Parasitic diseases</topic><topic>Protozoa</topic><topic>Protozoal diseases</topic><topic>Spectrophotometry</topic><topic>Subcellular compartmentation</topic><topic>Tropical medicine</topic><topic>Trypanosoma brucei</topic><topic>Trypanosoma brucei brucei</topic><topic>Trypanosoma brucei brucei - enzymology</topic><topic>Trypanosoma brucei brucei - ultrastructure</topic><topic>Trypanosomiasis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Aman, Rashid A.</creatorcontrib><creatorcontrib>Wang, Ching C.</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular and biochemical parasitology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Aman, Rashid A.</au><au>Wang, Ching C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Absence of substrate channeling in the glycosome of Trypanosoma brucei</atitle><jtitle>Molecular and biochemical parasitology</jtitle><addtitle>Mol Biochem Parasitol</addtitle><date>1986-04-01</date><risdate>1986</risdate><volume>19</volume><issue>1</issue><spage>1</spage><epage>10</epage><pages>1-10</pages><issn>0166-6851</issn><eissn>1872-9428</eissn><coden>MBIPDP</coden><abstract>Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966–6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of α-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of α-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5–5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form.</abstract><cop>Shannon</cop><pub>Elsevier B.V</pub><pmid>3012332</pmid><doi>10.1016/0166-6851(86)90059-9</doi><tpages>10</tpages></addata></record>
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ispartof Molecular and biochemical parasitology, 1986-04, Vol.19 (1), p.1-10
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source MEDLINE; Elsevier ScienceDirect Journals
subjects 2-Deoxyglucose
Adenosine Triphosphate - metabolism
Animals
Biochemistry. Physiology. Immunology. Molecular biology
Biological and medical sciences
Chromatography, High Pressure Liquid
Cross-Linking Reagents
Deoxyglucose - metabolism
Dimethyl Suberimidate
enzymes
Fructose-Bisphosphatase - metabolism
Fructosediphosphates - metabolism
Fundamental and applied biological sciences. Psychology
Glucose - metabolism
Glycerophosphates - metabolism
Glycolysis
Glycolytic enzymes
Human protozoal diseases
Infectious diseases
Medical sciences
Microbodies
Microbodies - enzymology
Multienzyme complex
Multienzyme Complexes - metabolism
NAD - metabolism
Oxidation-Reduction
Parasitic diseases
Protozoa
Protozoal diseases
Spectrophotometry
Subcellular compartmentation
Tropical medicine
Trypanosoma brucei
Trypanosoma brucei brucei
Trypanosoma brucei brucei - enzymology
Trypanosoma brucei brucei - ultrastructure
Trypanosomiasis
title Absence of substrate channeling in the glycosome of Trypanosoma brucei
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