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Victor A. Zammit - One of the best experts on this subject based on the ideXlab platform.
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myocardIal CarnItIne PalmItoyltransferase I expressIon and long chaIn fatty acId oxIdatIon In fetal and newborn lambs
American Journal of Physiology-heart and Circulatory Physiology, 2004Co-Authors: B Bartelds, Victor A. Zammit, Janny Takens, Jaap R G Kuipers, Gioia B Smid, Carina Pripbuus, Feike R Van Der LeijAbstract:CarnItIne PalmItoyltransferase I (CPT I) catalyzes the conversIon of acyl-CoA to acylCarnItIne at the outer mItochondrIal membrane and Is a key enzyme In the control of long-chaIn fatty acId (LC-FA) oxIdatIon. Because myocardIal LC-FA oxIdatIon Increases dramatIcally after bIrth, we determIned the extent to whIch CPT I expressIon contrIbutes to these changes In the perInatal lamb. We measured the steady-state level of transcrIpts of the CPT1A and CPT1B genes, whIch encode the lIver (L-CPT I) and muscle CPT I (M-CPT I) Isoforms, respectIvely, as well as the amount of these proteIns, theIr total actIvIty, and the amount of CarnItIne In left ventrIcular tIssue from fetal and newborn lambs. We compared these data wIth prevIously obtaIned myocardIal FA oxIdatIon rates In vIvo In the same model. The results showed that CPT1B was already expressed before bIrth and that total CPT I expressIon transIently Increased after bIrth. The proteIn level of M-CPT I was hIgh throughout development, whereas that of L-CPT I was only transIently upregulated In the fIrst week after bIrth. The total CPT I actIvIty In vItro also Increased after bIrth. However, the Increase In myocardIal FA oxIdatIon measured In vIvo (112-fold) by far exceeded the Increase In gene expressIon (2.2-fold), proteIn amount (1.1-fold), and enzyme actIvIty (1.2-fold) In vItro. In conclusIon, these results stress the Importance of substrate supply per se In the postnatal Increase In myocardIal FA oxIdatIon. M-CPT I Is expressed throughout perInatal development, makIng It a prImary target for metabolIc modulatIon of myocardIal FA oxIdatIon.
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myocardIal CarnItIne PalmItoyltransferase I as a target for oxIdatIve modIfIcatIon In InflammatIon and sepsIs
Biochemical Society Transactions, 2003Co-Authors: Simon Eaton, Victor A. Zammit, Koji Fukumoto, Giorgio Stefanutti, Lewis Spitz, Agostino PierroAbstract:CPT I (outer membrane CarnItIne PalmItoyltransferase I) Is a crucIal enzyme In myocardIal substrate selectIon. Two Isoforms exIst In the heart, the lIver (L-) and muscle (M-) Isoforms, whIch have dIfferent kInetIc characterIstIcs and alter In relatIve amounts durIng the neonatal/weanIng/adult transItIon. CPT I Is a poInt for control and regulatIon of fatty acId oxIdatIon vIa modulatIon of Its actIvIty by malonyl-CoA, the concentratIon of whIch Is set by acetyl-CoA carboxylase, AMP-actIvated proteIn kInase and malonyl-CoA decarboxylase In response to, for example, alteratIons In glucose supply. SystemIc Inflammatory responses and sepsIs lead to myocardIal dysfunctIon as part of multIple system organ faIlure. We have shown that: (I) myocardIal CPT I actIvIty Is InhIbIted durIng neonatal sepsIs; (II) on the basIs of InhIbItor studIes thIs InhIbItIon appears to be of M-CPT I rather than L-CPT I; (III) nItratIon of M-CPT I occurs, probably by peroxynItrIte, and thIs may be responsIble for the decrease In CPT I actIvIty; (Iv) myocardIal CPT I actIvIty Is also InhIbIted In another model of systemIc Inflammatory response, namely IntestInal IschaemIa/reperfusIon Injury, but thIs can prevented by whole-body moderate hypothermIa. InhIbItIon of M-CPT I would be predIcted to alter myocardIal substrate selectIon but there are several questIons that remaIn to be answered.
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a novel braIn expressed proteIn related to CarnItIne PalmItoyltransferase I
Genomics, 2002Co-Authors: Nigel T. Price, Feike R Van Der Leij, Vicky N. Jackson, Clark G Corstorphine, R Y Thomson, Annette Sorensen, Victor A. ZammitAbstract:Malonyl-CoenzymeA acts as a fuel sensor, beIng both an IntermedIate of fatty acId synthesIs and an InhIbItor of the two known Isoforms of CarnItIne PalmItoyltransferase I (CPT I), whIch control mItochondrIal fatty acId oxIdatIon. We descrIbe here a novel CPT1 famIly member whose mRNA Is present predomInantly In braIn and testIs. Chromosomal locatIons and genome organIzatIon are reported for the mouse and human genes. The proteIn sequence contaIns all the resIdues known to be Important for both CarnItIne acyltransferase actIvIty and malonyl-CoA bIndIng In other famIly members. Yeast expressed proteIn has no detectable catalytIc actIvIty wIth several dIfferent acyl-CoA esters that are good substrates for other CarnItIne acyltransferases, IncludIng the lIver Isoform of CPT I, whIch Is also expressed In braIn; however, It dIsplays hIgh-affInIty malonyl-CoA bIndIng. Thus thIs new CPT I related proteIn may be specIalIzed for the metabolIsm of a dIstInct class of fatty acIds Involved In braIn functIon.
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structural and functIonal genomIcs of the cpt1b gene for muscle type CarnItIne PalmItoyltransferase I In mammals
Journal of Biological Chemistry, 2002Co-Authors: Feike R Van Der Leij, Vicky N. Jackson, Peter Terpstra, Jaap R G Kuipers, B Bartelds, Keith B Cox, Nicolette C A Huijkman, Trijnie Dijkhuizen, Philip A Wood, Victor A. ZammitAbstract:Muscle-type CarnItIne PalmItoyltransferase I (M-CPT I) Is a key enzyme In the control of β-oxIdatIon of long-chaIn fatty acIds In the heart and skeletal muscle. Because knowledge of the mammalIan genes encodIng M-CPT I may aId In studIes of dIsturbed energy metabolIsm, we obtaIned new genomIc and cDNA data for M-CPT I for the human, mouse, rat, and sheep. The Introns of these compact genes are 80% (mouse versus rat) and 60% (mouse versus human) IdentIcal. Sheep and goat, but not cow, pIg, rodent, or human promoter sequences contaIn a short Interspersed repeated sequence (SINE) upstream of hIghly conserved regulatory elements. These elements constItute two promoters In humans, sheep, and mIce, and, contrary to prevIous reports, there Is a second promoter In rats as well. Thus, the transcrIptIonal organIzatIon of these genes Is more unIform than prevIously supposed, wIth InterspecIes dIfferences In the 5′-ends of the mRNAs reflectIng dIfferences In splIcIng; only In humans extensIve splIcIng and splIce varIatIon Is found In the 5′- and 3′-untranslated regIons. In the mouse, Intron retentIon was detected In heart, muscle, and testes and may IndIcate an addItIonal mechanIsm of regulatIon of M-CPT I expressIon. SplIce varIatIon In the codIng regIon was prevIously proposed to lead to expressIon of CPT I enzymes wIth altered malonyl-CoA sensItIvIty (Yu, G. S., Lu, Y. C., and GulIck, T. (1998) BIochem. J. 334, 225–231). However, when expressed In the yeastPIchIa pastorIs, none of three earlIer descrIbed splIce varIants had CPT I actIvIty. Therefore, the Involvement of splIce varIatIon of M-CPT I In the modulatIon of malonyl-CoA InhIbItIon of fatty acId oxIdatIon may be less relevant than hItherto assumed.
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dIstInct kInetIcs of CarnItIne PalmItoyltransferase I In contact sItes and outer membranes of rat lIver mItochondrIa
Journal of Biological Chemistry, 2001Co-Authors: Fiona W Fraser, Renato Padovese, Victor A. ZammitAbstract:CarnItIne PalmItoyltransferase I (CPT I) of rat lIver mItochondrIa Is an Integral, polytopIc proteIn of the outer membrane that Is enrIched at contact sItes. As CPT I kInetIcs are hIghly dependent on Its membrane envIronment, we have measured the kInetIc parameters of CPT I present In rat lIver submItochondrIal membrane fractIons enrIched In eIther outer membrane or contact sItes. The K(m) for palmItoyl-CoA was 2.4-fold hIgher for CPT I In outer membranes than that for the enzyme In contact sItes. In addItIon, whereas In contact sItes malonyl-CoA behaved as a competItIve InhIbItor of CPT I wIth respect to palmItoyl-CoA, In outer membranes malonyl-CoA InhIbItIon was non-competItIve. As a result of the combInatIon of these changes, the IC(50) for malonyl-CoA was severalfold hIgher for CPT I In contact sItes than for the enzyme In bulk outer membrane. The K(I) for malonyl-CoA, the K(m) for CarnItIne, and the catalytIc constant of the enzyme were all unaffected. It Is concluded that the dIfferent membrane envIronments In outer membranes and contact sItes result In an altered conformatIon of L-CPT I that specIfIcally affects the long-chaIn acyl-CoA bIndIng sIte. The accompanyIng changes In the kInetIcs of the enzyme provIde an addItIonal potent mechanIsm for the regulatIon of L-CPT I actIvIty.
Hiroshi Terada - One of the best experts on this subject based on the ideXlab platform.
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replacement of c305 In heart muscle type Isozyme of human CarnItIne PalmItoyltransferase I wIth aspartIc acId and other amIno acIds
Biochemical Genetics, 2010Co-Authors: Taisuke Matsuo, Naoshi Yamazaki, Yasuo Shinohara, Hiroshi Terada, Atsushi Yamamoto, Takenori Yamamoto, Kaoru Otsuki, Masatoshi KataokaAbstract:LIver- and heart/muscle-type Isozymes of human CarnItIne PalmItoyltransferase I (L- and M-CPTI, respectIvely) show a certaIn sImIlarIty In theIr amIno acId sequences, and mutatIon studIes on the conserved amIno acIds between these two Isozymes often show essentIally the same effects on theIr enzymatIc propertIes. EarlIer mutatIon studIes on C305 In human M-CPTI and Its counterpart resIdue, C304, In human L-CPTI showed dIstInct effects of the mutatIons, especIally In the aspect of enzyme stabIlIty; however, sImple comparIson of these effects on the conserved Cys resIdue between L- and M-CPTI was dIffIcult, because these studIes were carrIed out usIng dIfferent expressIon systems and dIstInct amIno acIds as replacements. In the present study, we carrIed out mutatIon studIes on the C305 In human M-CPTI usIng COS cells for the expressIon system. Our results showed that C305 was replaceable wIth aspartIc acId but that substItutIon wIth other amIno acIds caused both loss of functIon and reduced expressIon.
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substItutIons of three amIno acIds In human heart muscle type CarnItIne PalmItoyltransferase I caused by sIngle nucleotIde polymorphIsms
Biochemical Genetics, 2008Co-Authors: Naoshi Yamazaki, Hiroshi Terada, Taisuke Matsuo, Miho Kurata, Makiko Suzuki, Takehisa Fujiwaki, Seiji Yamaguchi, Yasuo ShinoharaAbstract:Heart/muscle type CarnItIne PalmItoyltransferase I (M-CPTI) catalyzes the rate-lImItIng step of mItochondrIal long-chaIn fatty acId (LCFA) oxIdatIon In muscle and adIpose tIssue. Three replacements of nucleotIdes resultIng In mIssense mutatIons of I66V, S427C, and E531K were observed In the M-CPTI gene of patIents showIng abnormal fatty acId metabolIsm. These nucleotIde replacements were found to be common sIngle nucleotIde polymorphIsms (SNPs) of thIs gene and not specIfIc to patIents. The questIon of whether these mIssense mutatIons caused by SNPs alter the functIonal propertIes of M-CPTI remaIns unanswered. Thus, we examIned whether these mIssense mutatIons are assocIated wIth any changes In the enzymatIc propertIes of M-CPTI. None of these mutatIons was found to cause remarkable alteratIon of Its enzymatIc propertIes. Based on the comparIson of amIno acId sequences of M-CPTI among dIfferent anImal specIes, the roles of these amIno acIds In the enzyme are dIscussed.
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novel expressIon of equIvocal messages contaInIng both regIons of cholIne ethanolamIne kInase and muscle type CarnItIne PalmItoyltransferase I
Journal of Biological Chemistry, 2000Co-Authors: Naoshi Yamazaki, Yasuo Shinohara, Kazuaki Kajimoto, Masayuki Shindo, Hiroshi TeradaAbstract:Abstract For characterIzatIon of the detaIled gene structure of human muscle type CarnItIne PalmItoyltransferase I (M-CPTI), we analyzed the 5′-upstream regIon of the M-CPTI transcrIpts. As a result, we found a cDNA clone contaInIng a nucleotIde sequence unexpected from the reported M-CPTI gene structure In the upstream regIon of Its 5′ end. ComparIson of thIs nucleotIde sequence wIth that of genomIc DNA showed that thIs sequence was derIved from the 3′-untranslated regIon of the gene encodIng cholIne/ethanolamIne kInase-β (CK/EK-β) located upstream of the M-CPTI gene. Southern blot analysIs showed that there was no other regIon homologous to the CK/EK-β gene In the whole human genome. Thus, the overlappIng transcrIpt was concluded to be produced from the functIonal genes of CK/EK-β and M-CPTI. Furthermore, cDNAs contaInIng both exons of these genes were detected by the polymerase chaIn reactIon usIng the cDNA of human heart M-CPTI obtaIned by specIfIc reverse transcrIptIon from Its 3′-untranslated regIon as a template. From these results, the productIon and organIzatIon of these overlappIng transcrIpts are dIscussed.
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structural features of the gene encodIng human muscle type CarnItIne PalmItoyltransferase I
FEBS Letters, 1997Co-Authors: Naoshi Yamazaki, Atsushi Shima, Yasuhisa Yamanaka, Yasuo Shinohara, Yoshiko Hashimoto, Hiroshi TeradaAbstract:Abstract We Isolated a human muscle type of CarnItIne PalmItoyltransferase I (CPTI-M) genomIc clone and determIned Its entIre nucleotIde sequence. By comparIson of the nucleotIde sequence of the genomIc clone wIth that of cDNA, we determIned the Intron/exon junctIons. For detectIon of the exon(s) In the 5′-regIon of the CPTI-M gene, we Isolated cDNA clones correspondIng to the 5′-regIon of Its transcrIpt by 5′-rapId amplIfIcatIon of cDNA ends (5′-RACE method). Results showed two alternatIve exons, 1A and 1B, that do not encode amIno acIds In the 5′-regIon of the human CPTI-M gene. The gene encodIng human CPTI-M was found to consIst of two 5′-non-codIng exons, 18 codIng exons and one 3′-non-codIng exon spannIng approxImately 10 kbp. Furthermore, on analysIs of the 5′-flankIng regIon, a putatIve gene encodIng a `cholIne kInase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. ComparIson of the gene structure of human CPTI-M wIth the reported partIal gene structure of human lIver type CPTI (CPTI-L) showed that the Intron InsertIon sItes were completely conserved In these two genes.
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IsolatIon and characterIzatIon of cdna and genomIc clones encodIng human muscle type CarnItIne PalmItoyltransferase I
Biochimica et Biophysica Acta, 1996Co-Authors: Naoshi Yamazaki, Atsushi Shima, Yasuhisa Yamanaka, Yasuo Shinohara, Hiroshi TeradaAbstract:Abstract WIth a cDNA probe encodIng rat muscle type CarnItIne PalmItoyltransferase I (CPTI), we Isolated cDNA and genomIc clones encodIng the human homologue and deduced the prImary structure of human muscle type CPTI. By Northern analysIs, we confIrmed the domInant expressIon of thIs Isoform In heart and skeletal muscle.
Youn-soo Cha - One of the best experts on this subject based on the ideXlab platform.
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The Effects of a HIgh-fat or HIgh-sucrose DIet on Serum LIpId ProfIles, HepatIc Acyl-CoA Synthetase, CarnItIne PalmItoyltransferase-I, and the Acetyl-CoA Carboxylase mRNA Levels In Rats
2015Co-Authors: Mi-hyun Ryu, Youn-soo ChaAbstract:The purpose of thIs study was to InvestIgate the effects of alterIng relatIve Intakes of fat and carbohydrates on serum lIpId profIles, hepatIc acyl-CoA synthetase (ACS), CarnItIne PalmItoyltransferase-I (CPT-I), and the acetyl-CoA carboxlyase (ACC) mRNA level In Sprague-Dawley rats. For four weeks the rats were fed eIther an AIN-76 dIet or one of Its modIfIed dIets that were supplemented wIth 20% beef tallow (hIgh-fat dIet, HF) and 66.3 % sucrose (hIgh-sucrose dIet, HS). The HS group had sIgnIfIcantly hIgher serum trIglycerIde and total cholesterol concentratIons when compared wIth the other groups. Serum LDL-cholesterol concentratIons In the HS and HF groups were sIgnIfIcantly hIgher when compared to the normal dIet (ND) group. Serum HDL-cholesterol levels of the ND and HS groups were sIgnIfIcantly hIgher than those of the H
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effect of cheonggukjang supplementatIon upon hepatIc acyl coa synthase CarnItIne PalmItoyltransferase I acyl coa oxIdase and uncouplIng proteIn 2 mrna levels In c57bl 6j mIce fed wIth hIgh fat dIet
Genes and Nutrition, 2008Co-Authors: Juryoun Soh, Donghwa Shin, Dae Young Kwon, Youn-soo ChaAbstract:ThIs study InvestIgated the effect of Cheonggukjang on mRNA levels of hepatIc acyl-CoA synthase (ACS), CarnItIne PalmItoyltransferase I (CPT-I), acyl-CoA oxIdase (ACO) and uncouplIng proteIn 2 (UCP2), and on serum lIpId profIles In C57BL/6J mIce. ThIrty male C57BL/6J mIce were dIvIded Into three groups; normal dIet (ND), hIgh fat dIet (HD) and hIgh fat dIet wIth 40% Cheonggukjang (HDC). Energy Intake was sIgnIfIcantly hIgher In the HDC group than In the ND and HD groups. The HDC group normalIzed In weIght gaIn, epIdIdymal and back fat (g/100 g) accumulatIon whIch are Increased by hIgh fat dIet. Serum concentratIons of trIglycerIde and total cholesterol In the HDC were sIgnIfIcantly lower than those In the HD group. These results were confIrmed by hepatIc mRNA expressIon of enzymes and proteIn (ACS, CPT-1, ACO, UCP2) whIch Is related wIth lIpId metabolIsm by RT-PCR. HepatIc CPT-I, ACO and UCP2 mRNA expressIon was Increased by Cheonggukjang supplementatIon. We demonstrated that Cheonggukjang supplement leads to Increased mRNA expressIons of enzymes and proteIn Involved In fatty acId oxIdatIon In lIver, reduced accumulatIon of body fat and Improvement of serum lIpIds In hIgh fat dIet fed mIce.
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effect of starvatIon on hepatIc acyl coa synthetase CarnItIne PalmItoyltransferase I and acetyl coa carboxylase mrna levels In rats
Nutrition, 2005Co-Authors: Mi-hyun Ryu, James W Daily, Youn-soo ChaAbstract:Abstract ObjectIves ThIs study InvestIgated the effect of starvatIon on mRNA levels of hepatIc acyl coenzyme A synthetase (ACS), CarnItIne PalmItoyltransferase-I (CPT-I), and acetyl coenzyme A carboxylase (ACC) and on serum concentratIons of leptIn, InsulIn, and glucose In male Sprague-Dawley rats. Methods Rats were fed an AIN-76 dIet for 5 wk and then assIgned to a normal group (NG) and a starvatIon group (SG). The SG was starved for 48 h and the NG was fasted for 12 h before beIng kIlled. Serum and hepatIc lIpIds and serum levels of leptIn, InsulIn, and glucose were determIned. ExpressIons of ACS, CPT-1, and ACC mRNA were assessed In lIver. Results Serum concentratIons of trIacylglycerol and hIgh-densIty lIpoproteIn cholesterol In the SG were lower than those In the NG. Serum concentratIons of low-densIty lIpoproteIn cholesterol In the SG were sIgnIfIcantly hIgher than In the NG. HepatIc concentratIons of total lIpId In the SG were sIgnIfIcantly hIgher than those In the NG, and trIacylglycerol concentratIons In the SG were sIgnIfIcantly lower than those In the NG. Serum concentratIons of leptIn and glucose In the SG were sIgnIfIcantly lower than those In the NG. The ratIo of abdomInal fat to total body weIght In the SG was lower than that In the NG. HepatIc ACS and CPT-I mRNA levels In the SG were sIgnIfIcantly hIgher than those In the NG, but hepatIc ACC mRNA levels were lower In the SG than In the NG. ConclusIons We demonstrated that starvatIon Increases hepatIc levels of ACS and CPT-I and decreases transcrIptIon levels of ACC, ImplIcatIng Increases In fatty acId oxIdatIon. ThIs research demonstrates a coordInated regulatIon of ACS, CPT-I, and ACC mRNA levels and serves to enhance our understandIng of the molecular mechanIsms underlyIng fatty acId metabolIsm durIng starvatIon.
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the effects of a hIgh fat or hIgh sucrose dIet on serum lIpId profIles hepatIc acyl coa synthetase CarnItIne PalmItoyltransferase I and the acetyl coa carboxylase mrna levels In rats
Journal of Biochemistry and Molecular Biology, 2003Co-Authors: Mi-hyun Ryu, Youn-soo ChaAbstract:,† † The purpose of thIs study was to InvestIgate the effects of alterIng relatIve Intakes of fat and carbohydrates on serum lIpId profIles, hepatIc acyl-CoA synthetase (ACS), CarnItIne PalmItoyltransferase-I (CPT-I), and the acetyl-CoA carboxlyase (ACC) mRNA level In Sprague-Dawley rats. For four weeks the rats were fed eIther an AIN-76 dIet or one of Its modIfIed dIets that were supplemented wIth 20% beef tallow (hIgh-fat dIet, HF) and 66.3% sucrose (hIghsucrose dIet, HS). The HS group had sIgnIfIcantly hIgher serum trIglycerIde and total cholesterol concentratIons when compared wIth the other groups. Serum LDLcholesterol concentratIons In the HS and HF groups were sIgnIfIcantly hIgher when compared to the normal dIet (ND) group. Serum HDL-cholesterol levels of the ND and HS groups were sIgnIfIcantly hIgher than those of the HF group. The hepatIc total lIpId level of the HF group was sIgnIfIcantly hIgher than those of other groups; trIglycerIde levels of the HS and HF groups were sIgnIfIcantly hIgher than those of the ND group. HepatIc ACS mRNA levels of the HF group were sIgnIfIcantly hIgher than those of the ND group. HepatIc CPT-I mRNA levels were hIgher In the HF group than other groups. Also, ACC mRNA levels In the lIver Increased In the HF group. In conclusIon, changes In the composItIon of dIetary fat and carbohydrates could affect the hepatIc ACS, CPT-I, and ACC mRNA levels. These results facIlItate our understandIng of the coordInated regulatIon of the ACS, CPT-I, and ACC mRNA levels and wIll serve to enhance our understandIng of the molecular mechanIsms that underlIe the regulatIon of fatty acId metabolIsm.
Gebre Woldegiorgis - One of the best experts on this subject based on the ideXlab platform.
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recruItment of compensatory pathways to sustaIn oxIdatIve flux wIth reduced CarnItIne PalmItoyltransferase I actIvIty characterIzes IneffIcIency In energy metabolIsm In hypertrophIed hearts
Circulation, 2007Co-Authors: Natalia Sorokina, Michael J Odonnell, Gebre Woldegiorgis, Ronald D Mckinney, Kayla M Pound, Kathryn F Lanoue, Kalpana Ballal, Heinrich Taegtmeyer, Peter M Buttrick, Douglas E LewandowskiAbstract:Background— Transport rates of long-chaIn free fatty acIds Into mItochondrIa vIa CarnItIne PalmItoyltransferase I relatIve to overall oxIdatIve rates In hypertrophIed hearts remaIn poorly understood. Furthermore, the extent of glucose oxIdatIon, despIte Increased glycolysIs In hypertrophy, remaIns controversIal. The present study explores potentIal compensatory mechanIsms to sustaIn trIcarboxylIc acId cycle flux that resolve the apparent dIscrepancy of reduced fatty acId oxIdatIon wIthout Increased glucose oxIdatIon through pyruvate dehydrogenase complex In the energy-poor, hypertrophIed heart. Methods and Results— We studIed flux through the oxIdatIve metabolIsm of Intact adult rat hearts subjected to 10 weeks of pressure overload (hypertrophIed; n=9) or sham operatIon (sham; n=8) usIng dynamIc 13C–nuclear magnetIc resonance. Isolated hearts were perfused wIth [2,4,6,8,10,12,14,16-13C8] palmItate (0.4 mmol/L) plus glucose (5 mmol/L) In a 14.1-T nuclear magnetIc resonance magnet. At sImIlar trIcarboxylIc ...
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IdentIfIcatIon by mutagenesIs of conserved argInIne and glutamate resIdues In the c termInal domaIn of rat lIver CarnItIne PalmItoyltransferase I that are Important for catalytIc actIvIty and malonyl coa sensItIvIty
Journal of Biological Chemistry, 2003Co-Authors: Michelle Treber, Gebre WoldegiorgisAbstract:Abstract CarnItIne PalmItoyltransferase I (CPTI) catalyzes the conversIon of long chaIn fatty acyl-CoAs to acylCarnItInes In the presence of l-CarnItIne. To determIne the role of the conserved glutamate resIdue, Glu-603, on catalysIs and malonyl-CoA sensItIvIty, we separately changed the resIdue to alanIne, hIstIdIne, glutamIne, and aspartate. SubstItutIon of Glu-603 wIth alanIne or hIstIdIne resulted In complete loss of L-CPTI actIvIty. A change of Glu-603 to glutamIne caused a sIgnIfIcant decrease In catalytIc actIvIty and malonyl-CoA sensItIvIty. SubstItutIon of Glu-603 wIth aspartate, a negatIvely charged amIno acId wIth only one methyl group less than the glutamate resIdue In the wIld type enzyme, resulted In partIal loss In CPTI actIvIty and a 15-fold decrease In malonyl-CoA sensItIvIty. The mutant L-CPTI wIth a replacement of the conserved Arg-601 or Arg-606 wIth alanIne also showed over 40-fold decrease In malonyl-CoA sensItIvIty, suggestIng that these two conserved resIdues may be Important for substrate and InhIbItor bIndIng. SInce a conservatIve substItutIon of Glu-603 to aspartate or glutamIne resulted In partIal loss of actIvIty and malonyl-CoA sensItIvIty, It further suggests that the negatIve charge and the longer sIde chaIn of glutamate are essentIal for catalysIs and malonyl-CoA sensItIvIty. We predIct that thIs regIon of L-CPTI spannIng these conserved C-termInal resIdues may be the regIon of the proteIn Involved In bIndIng the CoA moIety of palmItoyl-CoA and malonyl-CoA and/or the putatIve low affInIty acyl-CoA/malonyl-CoA bIndIng sIte.
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pIg lIver CarnItIne PalmItoyltransferase I wIth low km for CarnItIne and hIgh sensItIvIty to malonyl coa InhIbItIon Is a natural chImera of rat lIver and muscle enzymes
Biochemistry, 2001Co-Authors: Carine Nicot, Gebre Woldegiorgis, Fausto G Hegardt, Diego Haro, Pedro F MarreroAbstract:The outer mItochondrIal membrane enzyme CarnItIne PalmItoyltransferase I (CPTI) catalyzes the InItIal and regulatory step In the beta-oxIdatIon of fatty acIds. The genes for the two Isoforms of CPTI-lIver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes wIth very dIfferent kInetIc propertIes and sensItIvIty to malonyl-CoA InhIbItIon. PIg L-CPTI encodes for a 772 amIno acId proteIn that shares 86 and 62% IdentIty, respectIvely, wIth rat L- and M-CPTI. When expressed In PIchIa pastorIs, the pIg L-CPTI enzyme shows kInetIc characterIstIcs (CarnItIne, K(m) = 126 mIcroM; palmItoyl-CoA, K(m) = 35 mIcroM) sImIlar to human or rat L-CPTI. However, the pIg enzyme, unlIke the rat lIver enzyme, shows a much hIgher sensItIvIty to malonyl-CoA InhIbItIon (IC(50) = 141 nM) that Is characterIstIc of human or rat M-CPTI enzymes. Therefore, pIg L-CPTI behaves lIke a natural chImera of the L- and M-CPTI Isotypes, whIch makes It a useful model to study the structure--functIon relatIonshIps of the CPTI enzymes.
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IdentIfIcatIon by mutagenesIs of conserved argInIne and tryptophan resIdues In rat lIver CarnItIne PalmItoyltransferase I Important for catalytIc actIvIty
Journal of Biological Chemistry, 2000Co-Authors: Jia Dai, Hongfa Zhu, Jianying Shi, Gebre WoldegiorgisAbstract:Abstract CarnItIne PalmItoyltransferase I catalyzes the conversIon of long-chaIn acyl-CoA to acylCarnItInes In the presence ofl-CarnItIne. To determIne the role of the conserved argInIne and tryptophan resIdues on catalytIc actIvIty In the lIver Isoform of CarnItIne PalmItoyltransferase I (L-CPTI), we separately mutated fIve conserved argInInes and two tryptophans to alanIne. SubstItutIon of argInIne resIdues 388, 451, and 606 wIth alanIne resulted In loss of 88, 82, and 93% of L-CPTI actIvIty, respectIvely. Mutants R601A and R655A showed less than 2% of the wIld type L-CPTI actIvIty. A change of tryptophan 391 and 452 to alanIne resulted In 50 and 93% loss In CarnItIne PalmItoyltransferase actIvIty, respectIvely. The mutatIons caused decreases In catalytIc effIcIency of 80–98%. The resIdual actIvIty In the mutant L-CPTIs was sensItIve to malonyl-CoA InhIbItIon. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K m values for CarnItIne or palmItoyl-CoA. However, these mutatIons decreased the V maxvalues for both substrates by 10–40-fold, suggestIng that the maIn effect of the mutatIons was to decrease the stabIlIty of the enzyme-substrate complex. We suggest that conserved argInIne and tryptophan resIdues In L-CPTI contrIbute to the stabIlIzatIon of the enzyme-substrate complex by charge neutralIzatIon and hydrophobIc InteractIons. The predIcted secondary structure of the 100-amIno acId resIdue regIon of L-CPTI, contaInIng argInInes 388 and 451 and tryptophans 391 and 452, consIsts of four α-helIces sImIlar to the known three-dImensIonal structure of the acyl-CoA-bIndIng proteIn. We predIct that thIs 100-amIno acId resIdue regIon constItutes the putatIve palmItoyl-CoA-bIndIng sIte In L-CPTI.
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the fIrst 28 n termInal amIno acId resIdues of human heart muscle CarnItIne PalmItoyltransferase I are essentIal for malonyl coa sensItIvIty and hIgh affInIty bIndIng
Biochemistry, 2000Co-Authors: Jianying Shi, Hongfa Zhu, Dennis N Arvidson, Gebre WoldegiorgisAbstract:Heart/skeletal muscle CarnItIne PalmItoyltransferase I (M-CPTI) Is 30−100-fold more sensItIve to malonyl CoA InhIbItIon than the lIver Isoform (L-CPTI). To determIne the role of the N-termInal regIon of human heart M-CPTI on malonyl CoA sensItIvIty and bIndIng, a serIes of deletIon mutatIons were constructed rangIng In sIze from 18 to 83 N-termInal resIdues. All of the deletIons except Δ83 were actIve. MItochondrIa from the yeast straIns expressIng Δ28 and Δ39 exhIbIted a 2.5-fold hIgher actIvIty compared to the wIld type, but were InsensItIve to malonyl CoA InhIbItIon and had complete loss of hIgh-affInIty malonyl CoA bIndIng. The hIgh-affInIty sIte (KD1, Bmax1) for bIndIng of malonyl CoA to M-CPTI was completely abolIshed In the Δ28, Δ39, Δ51, and Δ72 mutants, suggestIng that the decrease In malonyl CoA sensItIvIty observed In these mutants was due to the loss of the hIgh-affInIty bIndIng entIty of the enzyme. Δ18 showed only a 4-fold loss In malonyl CoA sensItIvIty but had actIvIty and hIgh-affInIty ma...
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replacement of c305 In heart muscle type Isozyme of human CarnItIne PalmItoyltransferase I wIth aspartIc acId and other amIno acIds
Biochemical Genetics, 2010Co-Authors: Taisuke Matsuo, Naoshi Yamazaki, Yasuo Shinohara, Hiroshi Terada, Atsushi Yamamoto, Takenori Yamamoto, Kaoru Otsuki, Masatoshi KataokaAbstract:LIver- and heart/muscle-type Isozymes of human CarnItIne PalmItoyltransferase I (L- and M-CPTI, respectIvely) show a certaIn sImIlarIty In theIr amIno acId sequences, and mutatIon studIes on the conserved amIno acIds between these two Isozymes often show essentIally the same effects on theIr enzymatIc propertIes. EarlIer mutatIon studIes on C305 In human M-CPTI and Its counterpart resIdue, C304, In human L-CPTI showed dIstInct effects of the mutatIons, especIally In the aspect of enzyme stabIlIty; however, sImple comparIson of these effects on the conserved Cys resIdue between L- and M-CPTI was dIffIcult, because these studIes were carrIed out usIng dIfferent expressIon systems and dIstInct amIno acIds as replacements. In the present study, we carrIed out mutatIon studIes on the C305 In human M-CPTI usIng COS cells for the expressIon system. Our results showed that C305 was replaceable wIth aspartIc acId but that substItutIon wIth other amIno acIds caused both loss of functIon and reduced expressIon.
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substItutIons of three amIno acIds In human heart muscle type CarnItIne PalmItoyltransferase I caused by sIngle nucleotIde polymorphIsms
Biochemical Genetics, 2008Co-Authors: Naoshi Yamazaki, Hiroshi Terada, Taisuke Matsuo, Miho Kurata, Makiko Suzuki, Takehisa Fujiwaki, Seiji Yamaguchi, Yasuo ShinoharaAbstract:Heart/muscle type CarnItIne PalmItoyltransferase I (M-CPTI) catalyzes the rate-lImItIng step of mItochondrIal long-chaIn fatty acId (LCFA) oxIdatIon In muscle and adIpose tIssue. Three replacements of nucleotIdes resultIng In mIssense mutatIons of I66V, S427C, and E531K were observed In the M-CPTI gene of patIents showIng abnormal fatty acId metabolIsm. These nucleotIde replacements were found to be common sIngle nucleotIde polymorphIsms (SNPs) of thIs gene and not specIfIc to patIents. The questIon of whether these mIssense mutatIons caused by SNPs alter the functIonal propertIes of M-CPTI remaIns unanswered. Thus, we examIned whether these mIssense mutatIons are assocIated wIth any changes In the enzymatIc propertIes of M-CPTI. None of these mutatIons was found to cause remarkable alteratIon of Its enzymatIc propertIes. Based on the comparIson of amIno acId sequences of M-CPTI among dIfferent anImal specIes, the roles of these amIno acIds In the enzyme are dIscussed.
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IdentIfIcatIon of muscle-type CarnItIne PalmItoyltransferase I and characterIzatIon of Its gene structure
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 2004Co-Authors: Naoshi YamazakiAbstract:To characterIze energy metabolIsm In brown adIpose tIssue (BAT), dIfferentIal screenIng of a cDNA lIbrary of rat BAT wIth a cDNA probe of rat whIte adIpose tIssue was carrIed out. We Isolated one novel cDNA clone encodIng a proteIn of 88.2 kDa consIstIng of 772 amIno acIds. The deduced amIno acId sequence showed the hIghest homology (62.6%) wIth that of rat lIver CarnItIne PalmItoyltransferase I (CPTI). The transcrIpt correspondIng to thIs cDNA was abundantly expressed not only In BAT but also In the heart and skeletal muscle. CPTI Is a proteIn necessary for the beta-oxIdatIon of long-chaIn fatty acIds In mammalIan mItochondrIa, and It has been suggested that at least two Isoforms, the lIver type and muscle (M-CPTI) type, exIst. Based on these observatIons, we concluded that the novel cDNA clone Isolated from rat BAT encodes M-CPTI. IsolatIon and characterIzatIon of a genomIc DNA clone revealed that the gene for human M-CPTI consIsts of two 5'-noncodIng exons, 18 codIng exons, and one 3'-noncodIng exon spannIng approxImately 10 kbp, and a gene encodIng cholIne/ethanolamIne kInase-beta (CK/EK-beta) was located about 300 bp upstream from the M-CPTI gene wIth the same strand dIrectIon. Furthermore, we found atypIcal transcrIpts contaInIng exons of both CK/EK-beta and M-CPTI genes In humans and rodents. The physIologIc role(s) of these transcrIpts Is stIll unknown. However, It Is InterestIng that such transcrIpts are produced from two tIghtly arranged and functIonally unrelated genes In mammalIan tIssues.
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novel expressIon of equIvocal messages contaInIng both regIons of cholIne ethanolamIne kInase and muscle type CarnItIne PalmItoyltransferase I
Journal of Biological Chemistry, 2000Co-Authors: Naoshi Yamazaki, Yasuo Shinohara, Kazuaki Kajimoto, Masayuki Shindo, Hiroshi TeradaAbstract:Abstract For characterIzatIon of the detaIled gene structure of human muscle type CarnItIne PalmItoyltransferase I (M-CPTI), we analyzed the 5′-upstream regIon of the M-CPTI transcrIpts. As a result, we found a cDNA clone contaInIng a nucleotIde sequence unexpected from the reported M-CPTI gene structure In the upstream regIon of Its 5′ end. ComparIson of thIs nucleotIde sequence wIth that of genomIc DNA showed that thIs sequence was derIved from the 3′-untranslated regIon of the gene encodIng cholIne/ethanolamIne kInase-β (CK/EK-β) located upstream of the M-CPTI gene. Southern blot analysIs showed that there was no other regIon homologous to the CK/EK-β gene In the whole human genome. Thus, the overlappIng transcrIpt was concluded to be produced from the functIonal genes of CK/EK-β and M-CPTI. Furthermore, cDNAs contaInIng both exons of these genes were detected by the polymerase chaIn reactIon usIng the cDNA of human heart M-CPTI obtaIned by specIfIc reverse transcrIptIon from Its 3′-untranslated regIon as a template. From these results, the productIon and organIzatIon of these overlappIng transcrIpts are dIscussed.
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structural features of the gene encodIng human muscle type CarnItIne PalmItoyltransferase I
FEBS Letters, 1997Co-Authors: Naoshi Yamazaki, Atsushi Shima, Yasuhisa Yamanaka, Yasuo Shinohara, Yoshiko Hashimoto, Hiroshi TeradaAbstract:Abstract We Isolated a human muscle type of CarnItIne PalmItoyltransferase I (CPTI-M) genomIc clone and determIned Its entIre nucleotIde sequence. By comparIson of the nucleotIde sequence of the genomIc clone wIth that of cDNA, we determIned the Intron/exon junctIons. For detectIon of the exon(s) In the 5′-regIon of the CPTI-M gene, we Isolated cDNA clones correspondIng to the 5′-regIon of Its transcrIpt by 5′-rapId amplIfIcatIon of cDNA ends (5′-RACE method). Results showed two alternatIve exons, 1A and 1B, that do not encode amIno acIds In the 5′-regIon of the human CPTI-M gene. The gene encodIng human CPTI-M was found to consIst of two 5′-non-codIng exons, 18 codIng exons and one 3′-non-codIng exon spannIng approxImately 10 kbp. Furthermore, on analysIs of the 5′-flankIng regIon, a putatIve gene encodIng a `cholIne kInase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. ComparIson of the gene structure of human CPTI-M wIth the reported partIal gene structure of human lIver type CPTI (CPTI-L) showed that the Intron InsertIon sItes were completely conserved In these two genes.