Download 46 HL60 nuclei lacking the nuclear double membrane contain a

S590 Biochemical Society Transactions (1997) 25
46
HL60 nuclei lacking the nuclear double membrane
contain a PLD activity which is insensitive to the
ADP-ribosylationfactor.
JOANNA M. CLARK, MATTHEW N. HODGKIN and
MICHAEL J.O. WAKELAM
Figure 1. PLD activitv in HL60 nuclei which lack th e
puclear double membrane
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CRC Institute for Cancer Studies, Clinical Research Block,
University of Birmingham, Birmingham B15 2TA, U.K.
The possibility of a nuclear phospholipase D (PLD) has been
raised by at least two recent pieces of research. Firstly, nuclei
extracted from agonist-stimulated IIC9 fibroblasts contained
diradylglycerols which were probably derived from the hydrolysis
of phosphatidylcholine, the substrate of PLD [l]. Secondly, a
PLD activity has been described in nuclei of Madin-Darby canine
kidney cells[2]. In both instances, nuclei were prepared in the
absence of detergent. Nuclei generated in this way may be
contaminated with membranes contiguous with the outer nuclear
membrane, such as the endoplasmic reticulum (ER). In such a
preparation, a perinuclear enzyme activity could not be
distinguished from an intranuclear one. Nuclei prepared using
detergent lack the outer nuclear membrane. Thus, any enzyme
activity they contain is intranuclear.
The aim of this study was to determine whether
undifferentiated human myelomonocytic leukaemia (HL60) cells
have an intranuclear or a perinuclear PLD activity. HL60 cells
contain a high basal PLD activity, both in the cytosolic and in the
particulate fraction, which can be detected by the modified [3] in
vitro assay of Brown e f al. [4]. The particulate fraction PLD is
stimulated in vitro by either recombinant, myristoylated ADPribosylation factor 1 (rARFI) or HL60 cytosol, and stimulation by
these activators together is less than additive. Two methods were
used to extract the nuclei from HL60 cells. The extraction
method of Bunce ef al.[5] employed non-ionic detergent in order
to remove the nuclear membranes. That of Balboa et al. was
detergent-free and therefore likely to leave nuclear membranes
intact. Nuclei were assayed for PLD activity in vitro and were
screened for contamination by non-nuclear membranes.
HL60s cells were grown to a cell density of 106/ml in
RPMI 1640 with Glutamax-lTM,supplemented with 15% heatinactivated foetal calf serum and penicillin 100u/ml plus
streptomycin 100 pg/ml. Typically, 5 x lo8 cells were used for
each preparation. The nuclei were extracted by one or other of
the published methods referred to, but with the following changes:
in both cases, cell lysate equivalent to 5 x 107 cells in 500 pl was
loaded onto 500 ~ 1 5 0 %
sucrose (w/v) in a lysis buffer compatible
with the in vifro PLD assay (NaCI 137mM, Na2HP04 8.lmM,
KCI 2.7mM, KH2P04 1.5mM, EDTA 2.5mM pH 7.5 containing
dithiothreitol l m M , benzamidine
10pM
and
phenylmethanesulphonyl fluoride 200pM) and this buffer was
used at all subsequent stages of preparation. Nuclei from
detergent-treated cells were washed twice in this buffer to remove
the detergent, which interferes with the PLD assay. The final
nuclear preparation was a suspension of 5 x 108 cell
equivalentshl. Half of this suspension was disrupted by probe
sonication and the protein concentration determined. The
recovery of nuclei was between 1% - 15% of starting cell number
and the protein concentrations were in the order of 5mg/ml.
Preparations were stored overnight at -8O'C and assayed for PLD
activity the following day. They were screened for the presence
of membrane marker proteins: acid p -D-galactosidase
(lysosomes) [6], galactosyltransferase (golgi) [7] and glucoserelated protein grp78 (ER).
HL60 nuclei prepared using detergent contained a PLD
"
-0.5
Mean +/- range of duplicate determinations for one
nuclear preparation. PC hydrolysis due to the control
incubations (+/- cytosol and rARF1) have been subtracted.
The data was similar for two other preparations.
Table 1.
m
e
Lysosome
Golgi
ER
. . y (+/-) -r
t
M b t e i n N
P-D-galactosidase
0.9+/-1.6
galactosyltransferase 13+/-5
G1p78
u
c
163+/-117
96+/-80
+++
3
activity which was not activated by the assay constituents nor by
rARFl alone, but was activated by cytosol. rARFl showed
synergy with cytosol in stimulating this PLD. Western blotting
for grp78 and enzyme assay for P-D-galactosidase and
galactosyltransferase activities, showed the nuclei to be free of
contamination by other membranes. The absence of the nuclear
double membrane was confirmed by electron microscopy. Nuclei
prepared by the detergent-free method contained PLD activity
which was sensitive to both rARFl and HL60 cytosol. The
activity was thus similar to that seen in the HL60 whole
particulate fraction. However, these preparations were
contaminated with the lysosomal, golgi and endoplasmic
reticulum membrane marker proteins. Electron microscopy
showed the presence of the nuclear double membrane and
contiguous cytoplasmic structures. In addition, there was
contamination with whole cells.
In conclusion, HL60 cells show a low level of intranuclear
PLD activity. This activity is ARF-insensitive but is rendered
sensitive to activation by rARFl by a component of HL60
cytosol.
With thanks to LIZ DEACON for teaching nuclear extraction techniques
1. Jarpe, M.B., Leach, K.L. and Raben, D.M. (1994) Biochemistry 33,526-534
2. Balboa, M.A., Balsinde, J., Dennis, E.A. and Insel, P. (1995) J. Biol. Chem.
270 (20), 11738-11740
3. Martin, A., Brown, F.D., Hodgkin, M.N., Bradwell, A.J., Cook, S.J., Hart,
M.. and Wakelam, M.J.O. (1996) I. Biol. Chem. 271 (29), 17397-17403
4. Brown, A.H., Gutowski, S., Moomaw, C.R., Slaughter, C. and Sternweis, P.
(1993) Cell 75,1137-1 144
5. Bunce, C.M., Thick, J.A., Thick, Lord, J.M., Mills, D. and Brown, G.(1988)
Anal. Biochem. 175,67-73
6. Hayworth, C.M., Neuman, E.F. and Wyn, C.H. (1981) Biochem. J. 193,773779
7. Bretz, R. and Staubli, W. (1977) Eur. J. Biochem. 77, 181-192
Abbreviations: PLD, phospholipase D; rARFl, recombinant, myristoylated
ADP-ribosylation factor 1; ER, endoplasmic reticulum; Grp, glucose-regulated
protein; PC, phosphatidylcholine.
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