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Vol. 233. No. 1, 1997
TABLE 1
Activities ofPizospizogiucoisomerase (PGI), Gatalase, Faíty
Acid Syníhase (FAS), arad Acetyl-CoA Carbozyiase (AGC) ira
Post-Nuclear Supernataral (PNS) arad in Isolated Peroxisornes
from Livor of Control arad Di(2-ethylhexyl)phtaiate
(DEHP)-Treated Rata
Erazvnie
Catalase
PGI
FAS
ACC
PNS Peroxisonies
Coratrol DEHP
(gmol/min/mg proteira)
0.83 0.97
0.55 0.39
(omol/min/mg proteja)
1.88 0.11 1.14 = 0.03
0.34 t 0.03 0.42 0.01
Control DEHP
10.6
0.013
rad.
raM.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6.64
0.023
rad.
rad.
Note. PGI and catainse are marker enzymes for cytosol arad peroxisornes,
reapectiveiy. Data roproaerat mearas of two control arad two
DEHP-treated aninials. nd., noradetectable.
Hapatie peroxisames axbibil GPTactivity thaI is sensitive
lo inhibilion by malorayl-CoA (3, 25). To study a
poasible aaaociation ofAGG with Ibis organelle, peroxisornes
wore isolaled from liven liaaue oblainad from
rata treated with di(2-olhylbexyl)pbíalate (DEll?), a
proliferator afilie peroxisomal comparlmení (20). The
data ira Table 1 indicale Ihat Ibera is no associalian of
ACG aclivily witb peroxisomea from both control sud
DEHP-lraated aniniala. Likewise, no signiñcaní asaaciahora
of AGG lo peroxisomes was obsanved ora Ihe basia
of measunemerata of enzyxna masa (dala nol sbawn).
Qur receral abservation Ibal cyloakeletal compananta
are mosí likely involved ira Ihe control of Ihe activily
of GPT-I (26) prompted us lo corasider a cyloskelelal
localizaliora of ACG, since the latían erazyme is pivolal
ira Iha conírol of CPT-I activity (1). To sualyae a polential
aasociatiora of AGG witb Iba cytoskeleíon, isolaled
hepatocylaa ware ineubaled wilh vanioua compaunda
knowra lo affect cyloakelatal integrily. Subaequenlly,
celis were permeabilized by shorl-term trealmeral wilh
digilorain followed by rapid removal of released eytosalic
proteiras iracluding ACG. Ira Ibis approacb, AGG
asaoeiated with a aubeellular síruelune will be sedimeníed.
Tbe resulting pellel waa used lo detennine
AGG masa. The poasibilily of ara aaaacialiora betweora
ACO arad cytoakeletal comporaenta was teslad by the
use of okadaic acid (OA), taxol arad colchieine. QA has
beera ahown to disrupí Ihe cytoskelelora of hepalocytos
(27). Taxol birada lo lubulira arad síabilizea microtnbules,
preveraíirag Iba diaaaaembly of microlubulea ira a
veryefficieralfasbiora (28). Tbe polymaric atate of microtubules
can be dissociated by colehicine (29). Tbe data
of laMe 2 shaw thaI incubatiora of bepalocytea wilb OA
255
resulted ira Ibe release of substaratially more AGC tbsu
ira control colla. Interestiiigly, Iba OA-iraduced effecl
was eomplalely abolisbed by pretraating Iba hepalocytea
wilh taxol (Tabla 2). Likewiaa, prelreatmeral of
tha colla wilh KN-62, a specific inhibitor of Ca 2t’calmodulin-deperaderal
proleira binase II, also preveralad
Ihe OA-iraduced releasa of hapatie AGO. 5-Aminoimidazole-4-carboxamide
ribonucleaside (AlGAR), a spaciñc
activalar of5’-AMP-activaíed prolain binase, was wilhout
effect on Ihe retaralion of AGG maas ira celí gbosls
(Tabla 2). Incubaliora of he~alocytes witb colehicine did
nol affecl tbe release ofACC from digilorain-permeabilized
cella ajíhar. The dala obtained witb amyloglucosidase
(Tabla 2) suggeat thaI glycogan granulas nepresoral
a subeellular síructure caSable of binding AGG. To determina
whelher Ibe two AGG isofonma will diffareratially
release upan permeabilization, Ihe ceil gbosts
wene alao analyaad for Ihe presence of ACC-265 sud
AGG-280. However, irrespeclive of Iba incubaliora condition,
Iba ratio ofAGC-265/AGG-280 was identical, j.c.
AGG-280 was always about ana third of Ihe total ACO
masa (dala nol ahown).
DISCUSSION
Saveral aludies perfornied by albera (6-10) as well as
by our group (11) lod lo Ibe auggastion of aasocialion of
TABLE 2
Mass Measuremerata of Acetyl-GoA Carboxylase Relamed
ira Digitorain-Penneabiized Ral Hepalocyles
Additioras
Perceratage of total ACO masa
retairaed ira ce11 ghosts
Control 53.2 ±18.0
OA 20.6 ±8.0*
Taxol + OA 55.5 ±6.6
KN-62 -4- (JA 48.5 z 10.5
AICAR 46.0 ±19.5
Colchicine 64.5 8.7
Taxol + colchicine 67.9 ±1.0
Control’ 15.8 ±2.8
Control -4- amyloglucosidase’ 4.6 ±1.0*
Note. Hepatocytes were prein¿ibated for 20 mira with or without
10 pM taxol or 30 gM KN-62. incubadoras were coratinaed for 15
additional mira with or without 0.5 mM 5-amiraoimidazoie-4-carboxamide
riboraucleoside (AiICAR), 0.5 gM okadaic acid (OA) or 0.1 mM
coichicine. Subsequently, ccli ghosts were prepared as described ira
Materiala arad Metizoda. ACC retained ira tize cefighosts was quanti-
Sed by avidin-based ELISA annlysis using as tize probirag aratihody
a primary antiaerum against rat-liver ACC (17).
‘mese resuits aro from two seta of ccii gizoste of control celia
resuspended arad iracubated for 15 ndditionai mira with or without 50
U amylogiucosidnse. Resulta represerat the mean 5.1). of3 different
hepatocyte preparatioras. Tize aniourat ofACC preserat ira intact hepatocytes
was set nt 100%. Values of OA are signiñcantiy differerat
(Pco.01) versas ita control using the Studerat 1 test. This also applies
to aznyloglucosidase (P.