liiiMIIIfl~UDliiiMIII~U - Biblioteca de la Universidad Complutense ...

More documents

Recommendations

Info

However, no effect of AlGAR, ZMP, or 5’-AMP ora CPT- 1 activity ira either of the twa systems coníd be detected (results raot showra>. VELASCO ET AL. Malonyl-CoA-dependent and .independent mechan¿sms are invol ved in tite AJCAR-induced stimulation of CPT-1? It has been showra that CPT-I is reversibly serasitized to malonyl-CoA inhibition by malonyl-CoA itself[reviewed ira Ref. (11)]. Thus, whera mitoehondíla are incubated ira the abserace or preserace of malorayl- CoA, the enzyme becomes less or more serasitive to irabibitiora, respectively (11). This effect is also observed ira whole-cell systems: incubatiora of isolated hepatocytes witb compounds thaI decrease intracellular malorayl- CoA leveis makes the enzyme acquire a relaxed coraformation which exhibits high activity arad low serasitivity to inhibitiora by malonyl-CoA ira a subsequerat assay (11). The opposite ensues whera hepatocytes are exposed to compounds that iracrease malonyl-CoA conceratration, i.e, Ihe enzyme acquires a tight coraformatiora with low activity arad enhaneed sensitivity to inhibitiora by malorayl-CoA (11). Because permeabilization of Ihe plasma membrane of Ihe hepatocyte leads lo a large dilutiora of cytosolic comporaents iracludirag malonyl-CoA (22), arad the transitiora between Ihe two different sensitivity states is very fasí at 37 0C (11), tbe coraformational state of CPT-I within the intaet hepatocyte is only retairaed for very short periods of time ira permeabilized celís [cf. Ref. (15)]. The permeabilized-hepatocyte system tbus allows monitorirag the conformational state of CPT-I when tbe assay of erazyme activity is performed at different times after permeabilization. We used Ihis experimental approach lo leal whether the stimulation of CPT-I observed ira AICAR-treated hepatocytes aetuaily iravolves a deerease of intracellular malonyl-CoA levela. As atated aboye, the AlGARiradueed atimulation of [‘4G]palmitate oxidatiora correlated well with the AICAR-iraduced stimulation of CPT- 1 whera erazyme activity was determiraed 20 s after permeabilizatiora of Ihe hepalocyte plasma membrane (Pig. 1). Ira control incubatioras, a lag phase ira Ihe CPT- 1 assay was observed at very short reaction times (Fig. 2). This lag phase seems tobe dueto the coraformational corastrainís of the CPT-I protein iraduced by intracellular malonyl-CoA [cf. Ref. (15)], arad it rapidly disappears after complete leakage of malorayl-CoA from the permeabilized ceils, allowing relaxation of the enzyme (Fig. 2). Ira contrast, by depletirag intracellular malorayl- CoA, ALGAR was indeed able to eliminate Ihe lag pbase inhererat to the permeabilized-ceil assay of CPT-I activity (Fig. 2). The magnitude of the AICAR-induced stimulation of CPT-I was reduced lo 45 t 14% at 40 s afler permeabilizatiora arad lo 30 t 12% at 60 s after permeabilization (Fig. 2), indicating thaI differences in the conformational state of the CPT-1 protein belweera con- o o. o o. o o s c -s e 2’— oa o o 60 Reaction time (s) ¡ ¡ 1 ¡ FIO. 2. Effact of AICAR on CP’I’-l activity at dilfororat reaction timos. Hepatocytes were proincubated for 15 mira ira tSe abseraco (O) or ira the preserace (e) of0.5 mM AICAR. Subsoqueratly, aliquots wOre removed to dotermirae CPT-I activity at difToront reaction times by method A. Noto that enzyme volocity (arad not product accurnulatiora) ‘a represerated ora tSey-axis. Results corresporad to six different Sopatocyte preparratioras. trol arad AICAR-treated celís are disappearing. However, a constaral 20—25% stimulatiora of CPT-I activity was observed ira MCAR-treated hepatocytes up to 140 a after hepatocyte permeabilizatiora (Fig. 2). Ira aur ayatem, this time is believed to ‘be more than eraough to allow reversal of coraformational corastrairata of CPT-I induced by charages ira intraceilular malarayl-CoA conceníratiora [cf. Ref. (11)]. To obtain more direcí evidence for the involvemeral of a malonyl-CoA-independent componení ira the AlGARiraduced atimulatiora of CPT-L erazyme aetivity was determiraed by a more complex procedure whicb eliminates any posaible interfereraceofmalorayl-CoA. Ira such ara assay (method B), hepalocytes are penneabilized with digitonira, followed by rapid arad extensive washíng of permeabilized celís to allaw complete remaval of malorayl-CoA. Thera, permeabilized celís are iracubated al 370C for up la 15 mira prior lo determinatiora of erazyme activity, so thaI any conformational carastrairal of the CPT-I enzyme will disappear (15). The use of tbis pracedure corroborated thaI a 20—25% atimulation of CPT-I by AlGAR is exerted by a malorayl-CaA-iradeperadení mecharaism (Fig. 3). Mareover, Ihe magrailude of tbe stimulaliora of CPT-I as delermiraed by method B was ideratical with reactiora times of 20, 60, arad 120 s (resulta nol shown). Note thaI for determinaban of CPT-I activity by method B cytosolie proteira that leaked from the permeabilized cella is removed prior to determinatiora of erazyme aetivity, arad so erazyme specific activity (referred lo as milligrama of prolein) waa always higher ira melbod B Ihan ira method A (note to Table 1). 120
-co 100 n H o ocoo E ci, Method 50 o’ ere CONTROL OF’ FArPY ACID OXIDATION BY AMP-ACTI7VATED PROTEIN tUNASE * * A 8 8(15> C D FIG. 3. Differential offect of AICAR on CPT-l activity as determineé by methods A, B, C, arad D. Hepatocytes wero preiracubated for 15 mira ira tho absence or ira tSe preserace of 0.5 m=¿AICAR. Subaequeratly, aliquots were removed to determine CPT-t activity by the various methoda. In tSe case of method B, the ghosts of permeabilized celia woro iracubated at Mt for Sor 15 mira prior to doterminatiora of enzyxrao activity. Roactiora time waa 20 a for method A arad 60 a for methods B, C, ané D. Resulta corresporad to eight differerat hepatocyte preparatioras. Significaratly difforerat vorsus incubatioras with rao additions: *~P c 0.05; **~P < 0.01. Malonyl-CoA-independent stimulation of CPT-I by AICA.R may involve interaction of CPT-Iwith cytoskeletal components. The obaervatiora thaI Ibe phospbatase irahibitor okadaic acid atimulates hepatie CPT-I by a stable mecharaism led lo Ihe auggeslion thaI irabibihora of Ihe pbosphalaaes might have resulled ira iracreased phosphorylation of CPT-I, with a corasequeral iracrease of erazyrrae aetivity (7). However, iracubalion of purified mitochoradrial auter membranes or isolated mitoehoradria with differerat proteira kiraases (iracluding TABLE 1 173 AMPK) arad protein phosphatases did nol lead to aray charage ira Ihe kiraetic arad regulatory properties of CPT- 1(15). It has also been showra thaI the malonyl-CoAiradeperadení iracrease of CPT-I activity observed ira okadaic acid-treated hepatoeytes is not due lo Ihe direcí phoaphorylátiora of Ihe erazyme, buí may involve modulation of interactioras between CPT-I arad raoradiffusible extramitochoradrial comporaerata (15). Three observationa indicate that Ibis may also be Ihe case of the malorayl-CoA-iradeperaderat componení ofIhe ALGAR-iraduced stimulaíiora of CPT-I:: (1) Stimulation of CPT-I was nol apparent when erazyme activity waa measured,ira mIad mitochoradria isolated from ALCAR-treated hepatocytes (meihod D), despile Ihe presence of 50 mM fluoride ira ah Ibe isolatiora buifera (Fig. 3). Likewise, when hepatocyles were vigoroualy disrupled by soraicatiora after AlGAR trealmení (methad G), Ihe stimulatory effect of ALGAR ora GPT-I waa nol preserved (Fig. 3). Thus, malorayl-GoA-independení stimulatiora ofCPT-I by ALGAR is oraly evideral whera mitochoradria are still assoeiated with olber cellular fractioras, le., ira Ihe ghosta of Ihe permeabilized celís, buí nol after separaliora of mitochondria from olber celí componerats. Therefore, it appeara thai the malonyl-GoA-iradeperaderat stimulatiora of CPT-I by AlGAR requires comporaenís of Ihe extramitocharadrial compartmerat of Ihe celí. (u) Tbe possibility Ihat cytoakeleíal comporaerais may be involved ira Ihe malorayl-GoA-iradeperadení aIimulatiora of CPT-I by AlGAR was leated by usirag taxol. This complex dilerpenoid binda lo tubulin arad atabilizos mierotubules, preveratirag the disaasembly of microtubules ira a very effieientfashion (25). Iraterestiragly, malorayl-GoA-iradeperaderat activatiora of CPT-I iraduced Combinod Effects of AICAR, Okadaic Acid. arad Taxol on the Activities of CP’I’-I and Acetyl-CoA Garboxylase Adéitiona CPT-I activity (ramol/minlmg protoira) Method A Method E Acetyl-CoA carboxylase activity
Page 1 and 2:
UNiVERSIDAD COMPLUTENSE DE MADRID F
Page 3 and 4:
1. INTRODUCCIÓN ÍNDICE 1.1. ASPEC
Page 5 and 6:
L Introducción
Page 7 and 8:
Canítulo 1 Introducción en forma
Page 9 and 10:
Can/tu/o ¡ 1988) y sales biliares
Page 11 and 12:
Capítulo 1 Introducción sistema d
Page 13 and 14:
Capitulo 1 Introducción ble la sí
Page 15 and 16:
Can¡tu/o 1 introducción Ca racten
Page 17 and 18:
Cadhulo 1 Introducción drial exter
Page 19 and 20:
Canítulo 1 Introduccián del malon
Page 21 and 22:
Capitulo ¡ Introducción Preincuba
Page 23 and 24:
Canítulo ¡ BIBLIOGRAFLA Abunirad,
Page 25 and 26: Capítulo ¡ Rodríguez, J.C., Cil-
Page 27 and 28: 2.1. Efectos deJATP extracelulary d
Page 29 and 30: Effects of extracellular ATP Qn hep
Page 31 and 32: Tabla 1. Effect of extracellular A=
Page 33 and 34: EFFECTS OF EXTRACEI 4LULARATP Di HE
Page 35 and 36: EFFECTS OF EXTRACELLTJLAR AIF Di HE
Page 37 and 38: 240 snspansions ware incubatad at 3
Page 39 and 40: Capitulo 2 Bibliograria. DISCUSIÓN
Page 41 and 42: Capitulo 2 INTRODUCCIÓN Resultados
Page 43 and 44: BIOCHEMICAL AND B!OPHYSTCAL RESEARC
Page 45 and 46: Vol. 224, No. 3, 1996 BIOCHEMICAL M
Page 47 and 48: Vol. 224. No. 3, 1996 . BIOCHEMICAL
Page 49 and 50: ajochen. J. (1997)321. 211—216 (P
Page 51 and 52: Table 1 Effect al protejo kinase in
Page 53 and 54: ab cd — — — FIgure 4 Eblect o
Page 55 and 56: THE JOURNAL OP WOLOGICAL CI4EM¡SIR
Page 57 and 58: Immunop~rcipi¡añon of 32P-¡abeil
Page 59 and 60: permeabilizod hepatocytos and CPT-I
Page 61 and 62: (autbors’ urapublisbad rosullis).
Page 63 and 64: TItE JOURNAL OF BIoLoGICA[. CIIEMIS
Page 65 and 66: 2.3. Posible papel fisiológico del
Page 67 and 68: LOSS OF RESPONSE OF CARNITINE PALMI
Page 69 and 70: Lowenstein, Brandeis Uraiversity, W
Page 71 and 72: TABLE 2. Comparative effect of okad
Page 73 and 74: ARCHIVES OF BIOCHEMJSTRY AND BIOPHY
Page 75: CONTROL OF FATTY ACID OXIDATION BY
Page 79 and 80: CONTROL OF FAflY ACID OXIDATION BY
Page 81 and 82: Vol. 233, No. 1. 1997 íonxc strera
Page 83 and 84: Vol. 233, No. 1, 1997 BIOCIjEMICAL
Page 85 and 86: Capítulo 2 DISCUSIÓN Relvultados
Page 87 and 88: Cavitulo .3 ¡3kcu~ión General y C
Page 89 and 90: Cacílulo 3 Esquema 2 >1. APOPTOSIS
Page 91: Canítulo 3 Discusión Géneral y C
show all

liiiMIIIfl~UDliiiMIII~U - Biblioteca de la Universidad Complutense ...

Create successful ePaper yourself

Delete template?

Save as template?