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fliE JOURNAI. OF WOWG¡CM. CHEMISTRY
HG. 1. FIlen of n.ild brypsin digestion en CPT-I actidby la
penneabilized hepatocytes. Hopatocytos were preincutiated for 15 mm ira
dio absonce (o) or ira Iba presoraco (@)of 0.5 gM OA. Ccli; ~cre ehen
pormoabilized váth digitorain arad thorougJy washod with 40 volumes cf
digitonin-free modium as described ira Experimental Procedures.
Permoabulized hopatocytes were subsequoratly resuspendod at 1.5-2.0 mg
protejo/ml and troated with vatying concentrations of tzypsin at 4C.
Trypsin action was stoppcd aftor 2 mira ti>’ additiora of 10 mg/ml of bovino
serum albuniin arad immodiate washing with 40 volumos of erypsin-froe
medium. mora. CPT-T activity was detormined ira thoso penneabilizod
hepatocytes. One-bundrod percoral CPT-i activity was 1.87±0.20
ramol/min ir mg protoira. Resu¡ts corrospond te 4 d¡fferont cali
preparation;. lnset: Mitochondria were isolatod from pormeabulized
hepatocytos that liad beco troatod wiLbout (lane a) or váth (lario ti) 17.5
gg/mI tzypsin, arad CPT-I was detected ti>’ Westorn blctting. ‘Dio arrow
points te iba 88-KDa band.
Effect of disn¿ptors of tite cytoskeleton oit CpT-i ac¡iviry -
OA and otber phosphataso inhibitors produce
hyperphosphorylarion and corasoquontly disruption of the
cytoskeletal network ira sevaral cail types, including
hopatocytes (e.g. 16,17). To test whether changas in Lho
organization of the cytoske¡eton may be related to parallel
changas ira CPT-I activity, hepatocytcs were incubaLod wath
colchicune (a microtubule disrupLor), cytochalasin B (a
m¡crofilamont disrupror), and IDPN (ara intermediatefilament
disruptor) (18,19). As shown in Table II, raeithor
coichicine nor cytochalasun B affected CPT-I acrivity. A; a
control to prova the biologica] activity of Lhese Nro
compounds on hepatocytes, cellular lipid; were pre-labeiled
with [‘
4C]palmitate, and very-low-derasity-lipoprotein output
anto the medium was monitorod (26). As previousiy
described (20), disruption of microtubules wirh colciiicine
or disruption of microfilamonts with cytochalasira 8 led to
a sLrong inhibition (>90%) of the output of very-lowdonsity-lipoprotoin
lipids into the medium arad to a parallal
accumulaLion of intracellular lipids.
»
TABLE II
Effces ofdin,,xas of¡he qeatkMon a. CPUn* .~ i0m4 frtei
¡
Hentccvtes were premoibetcd for 45 mira lo tite k — la ¡be ptn of tite
nnowaaon of cvnkcleuá inujmny indialsd. ¡aaÉs~ we,t anaS ter 15
adadonal — váab or “.,thout 0.5 »MOA. aaá din .Iiquoc .exe mkn te delanune
cfl”-i by tite one-etepsafas w.I¡ ma wuloa>4.CoA¡o.k Cw.4n~S pan van
of CFI-! actnwv andmalouiyl-CaAaeennnozwere ).29±ftflo,neI/mnnz mgpmtelfl
and 73±12pmoi/mg protein. respeaivciy. Rh. an~k