Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)
In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala ( Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA ox...
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description | In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala (
Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver
AOX cDNAs, designated
AOX1 and
AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala
AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala
AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent
K
m values for
AOX1 and
AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala
AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single
AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single
AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes. |
doi_str_mv | 10.1016/S0378-1119(03)00491-8 |
format | Article |
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Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver
AOX cDNAs, designated
AOX1 and
AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala
AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala
AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent
K
m values for
AOX1 and
AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala
AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single
AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single
AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes.</description><identifier>ISSN: 0378-1119</identifier><identifier>EISSN: 1879-0038</identifier><identifier>DOI: 10.1016/S0378-1119(03)00491-8</identifier><identifier>PMID: 12758125</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Acyl-CoA Oxidase ; Amino Acid Sequence ; Animals ; Base Sequence ; Blotting, Northern ; Cloning, Molecular ; COS Cells ; DNA, Complementary - chemistry ; DNA, Complementary - genetics ; Gene Expression Regulation, Enzymologic ; Humans ; Isoenzymes - genetics ; Liver - enzymology ; Marsupialia - genetics ; Molecular Sequence Data ; Oxidoreductases - genetics ; Oxidoreductases - metabolism ; Palmitoyl-coenzyme A ; Peroxisomal β-oxidation ; Peroxisomes - enzymology ; Rapid amplification of cDNA ends ; Rats ; Reverse transcription–polymerase chain reaction ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; Sequence Analysis, DNA</subject><ispartof>Gene, 2003-05, Vol.309 (2), p.91-99</ispartof><rights>2003 Elsevier Science B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c392t-5563f824552424969c6aae2ff0262798cd2d652317a5a1f2158457db0bbb4da3</citedby><cites>FETCH-LOGICAL-c392t-5563f824552424969c6aae2ff0262798cd2d652317a5a1f2158457db0bbb4da3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0378-1119(03)00491-8$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12758125$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ngo, Suong Ngoc Thi</creatorcontrib><creatorcontrib>McKinnon, Ross Allan</creatorcontrib><creatorcontrib>Stupans, Ieva</creatorcontrib><title>Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)</title><title>Gene</title><addtitle>Gene</addtitle><description>In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala (
Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver
AOX cDNAs, designated
AOX1 and
AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala
AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala
AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent
K
m values for
AOX1 and
AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala
AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single
AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single
AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes.</description><subject>Acyl-CoA Oxidase</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Blotting, Northern</subject><subject>Cloning, Molecular</subject><subject>COS Cells</subject><subject>DNA, Complementary - chemistry</subject><subject>DNA, Complementary - genetics</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>Humans</subject><subject>Isoenzymes - genetics</subject><subject>Liver - enzymology</subject><subject>Marsupialia - genetics</subject><subject>Molecular Sequence Data</subject><subject>Oxidoreductases - genetics</subject><subject>Oxidoreductases - metabolism</subject><subject>Palmitoyl-coenzyme A</subject><subject>Peroxisomal β-oxidation</subject><subject>Peroxisomes - enzymology</subject><subject>Rapid amplification of cDNA ends</subject><subject>Rats</subject><subject>Reverse transcription–polymerase chain reaction</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - metabolism</subject><subject>Sequence Analysis, DNA</subject><issn>0378-1119</issn><issn>1879-0038</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9P3DAQxa2qqGxpP0KRTxUcAv4TJ86pWq2gICGBVO6WY4-LixNvbS-wEh-eLLuCI3MZjfSbeaP3EPpByQkltDn9Q3grK0ppd0T4MSF1Ryv5Cc2obLuKEC4_o9kbso--5vyPTCUE-4L2KWuFpEzM0POlhbF4540uPo5YjxabEEc__sXR4fIYsYtpyJsh-AdIeAkpPvkcBx2w06Ws8dysA17EOb5-8lZnwC7FAZc7wPdRB42P8M2dziYGnUyJGRs_QoJVPv6G9pwOGb7v-gG6PT-7XVxUV9e_Lxfzq8rwjpVKiIY7yerp95rVXdOZRmtgzhHWsLaTxjLbCMZpq4WmjlEha9HanvR9X1vND9DP7dlliv9XkIsafDYQgh4hrrJqOZNNPYl8BFIpSS0kn0CxBU2KOSdwapn8oNNaUaI28ajXeNTGe0W4eo1HyWnvcCew6gew71u7PCbg1xaAyY4HD0ll42E0YH0CU5SN_gOJF88sn14</recordid><startdate>20030508</startdate><enddate>20030508</enddate><creator>Ngo, Suong Ngoc Thi</creator><creator>McKinnon, Ross Allan</creator><creator>Stupans, Ieva</creator><general>Elsevier B.V</general><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>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20030508</creationdate><title>Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)</title><author>Ngo, Suong Ngoc Thi ; McKinnon, Ross Allan ; Stupans, Ieva</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c392t-5563f824552424969c6aae2ff0262798cd2d652317a5a1f2158457db0bbb4da3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Acyl-CoA Oxidase</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Blotting, Northern</topic><topic>Cloning, Molecular</topic><topic>COS Cells</topic><topic>DNA, Complementary - chemistry</topic><topic>DNA, Complementary - genetics</topic><topic>Gene Expression Regulation, Enzymologic</topic><topic>Humans</topic><topic>Isoenzymes - genetics</topic><topic>Liver - enzymology</topic><topic>Marsupialia - genetics</topic><topic>Molecular Sequence Data</topic><topic>Oxidoreductases - genetics</topic><topic>Oxidoreductases - metabolism</topic><topic>Palmitoyl-coenzyme A</topic><topic>Peroxisomal β-oxidation</topic><topic>Peroxisomes - enzymology</topic><topic>Rapid amplification of cDNA ends</topic><topic>Rats</topic><topic>Reverse transcription–polymerase chain reaction</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - metabolism</topic><topic>Sequence Analysis, DNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ngo, Suong Ngoc Thi</creatorcontrib><creatorcontrib>McKinnon, Ross Allan</creatorcontrib><creatorcontrib>Stupans, Ieva</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Gene</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ngo, Suong Ngoc Thi</au><au>McKinnon, Ross Allan</au><au>Stupans, Ieva</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)</atitle><jtitle>Gene</jtitle><addtitle>Gene</addtitle><date>2003-05-08</date><risdate>2003</risdate><volume>309</volume><issue>2</issue><spage>91</spage><epage>99</epage><pages>91-99</pages><issn>0378-1119</issn><eissn>1879-0038</eissn><abstract>In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala (
Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver
AOX cDNAs, designated
AOX1 and
AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala
AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala
AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent
K
m values for
AOX1 and
AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala
AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single
AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single
AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>12758125</pmid><doi>10.1016/S0378-1119(03)00491-8</doi><tpages>9</tpages></addata></record> |
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subjects | Acyl-CoA Oxidase Amino Acid Sequence Animals Base Sequence Blotting, Northern Cloning, Molecular COS Cells DNA, Complementary - chemistry DNA, Complementary - genetics Gene Expression Regulation, Enzymologic Humans Isoenzymes - genetics Liver - enzymology Marsupialia - genetics Molecular Sequence Data Oxidoreductases - genetics Oxidoreductases - metabolism Palmitoyl-coenzyme A Peroxisomal β-oxidation Peroxisomes - enzymology Rapid amplification of cDNA ends Rats Reverse transcription–polymerase chain reaction RNA, Messenger - genetics RNA, Messenger - metabolism Sequence Analysis, DNA |
title | Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus) |
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