Abstract

1
Abstract
A Non-radioactive Assay to Determine Isoform
Activation of PLD by Phenylephrine in CCL39
Cells
Danielle E. Rastedt, Mark A. Wallert, and Joseph
J Provost Department of Biosciences Minnesota
State University Moorhead, Moorhead MN 56563
Methods
Phospholipase D (PLD) is an enzyme found in the
cells of higher mammals and plants. The process
of PLD acting on phosphatidylcholine (PC) to
produce phospatidic acid (PA) and choline is
important in cell signaling. PA acts as a
bioactive lipid activating a number of protein
kinases and other effectors and can be further
metabolized to diacylglycerol, an activator of
protein kinase C (PKC). There are two isoforms of
PLD, PLD1 and PLD2. PLD1 activity is activated
by the small G proteins RhoA and ARF as well as
PKC, while PLD2 is constitutively active and can
be stimulated by ARF. There is great interest in
understanding which isoforms is activated by
various hormones. Therefore, several different
methods have been developed to determine its
enzymatic activity. The current method used to
determine enzymatic activity is an in vivo PLD
assay using radioactive lipids. Our plan is to
use fluorescent labels to measure PLD activity in
a non-radioactive assay. The three types of
fluorescent lipids used in these experiments. Two
free fatty acids 4,4-difluoro-5-octyl-4-bora-3a,4
a-diaza-s-indacene-3-pentanoic acid (BODIPY C8,
C5) 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-in
da-cene-3-dodecanoic acid (BODIPY C1, C12) and
1-myristoyl-2-, 12-(7-nitro-2-1,
3-benzoxadiazol-4-yl)aminododecanoyl-sn-glycero-
3-phosphocholine (NBD-PC). We found that both
NBD-PC and BODIPY C1, C12 but not BODIPY C8, C5
were incorporated into the membrane as PC.
Furthermore, there is a dose and time dependent
manner in the labeling of starved CCL39
fibroblasts. We plan to show which PLD
isoforms(s) is activated by stress hormones in
CCL39 cells using this technique.
Results
Separation Solvent system- A separation of the
free fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a
-diaza-s-inda-cene-3-dodecanoic acid (BODIPY C1,
C12), 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-
s-indacene-3-dodecanoy0-1-hexadecanoyl-sn-glycero-
3-phosphocholine (?-BODIPY 500/510 C12-HPC),
2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-inda
cene-3-dodecanoy0-1-hexadecanoyl-sn-glycero-3-phos
phate, diammonium salt ((?-BODIPY 500/510 C5
HPA), and ptd BD BODIPY were tested. 15?l of
0.1?g/?l or 1?g/?l was applied to a thin layer
chromatography. Four different solvents were
tested. Solvent 1- ethyl acetate2,2,4 trimethyl
pentane acetic acid water (65101550 v/v).
Solvent 2- chloroform methanol water acetic
acid (4545101 v/v). Solvent 3- Chloroform
methanol water (63343 v/v). Solvent 4-
chloroform methanol water (454510 v/v). The
solvents were used to develop the TLC plate The
fluorescent lipids were detected by densitometry
using UV light on the Gel Doc 1000 (Bio-Rad).
Sensitivity of Visualization- Appropriate
amounts of 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-d
iaza-s-indacene-3-dodecanoy0-1-hexadecanoyl-sn-gly
cero-3-phosphate, diammonium salt ((?-BODIPY
500/510 C5 HPA)were applied to a thin layer
chromatography. The solvent used to develop the
TLC plate consisted of chloroform/methanol/water
(63343 v/v). The fluorescent lipids were
detected by densitometry using UV light on the
Gel Doc 1000 (Bio-Rad). A further analysis of the
detection of fluorescents was achieved by
scraping the silica from the TLC plate into
microfuge tube. The silica was rinsed with 0.3mL
of methanol and then vortex, centrifuged, and
incubated at room temperature for 10 minutes. 200
?l was extracted from the solution and measured
for fluorescents using the plate reader. Cell
Culture- Chinese hamster lung fibroblasts (CCL39)
were cultured in attachment culture using
Dulbeccos modified Eagle medium (DMEM, Sigma)
and 10 heat-activated fetal bovine serum (FBS,
GibocBRL) 37oC in a 5 CO2 incubator. Cells were
allowed to grow to 60 confluence in 35mm dishes.
Appropriate amounts of resuspended lipids were
added to each dish, and were allowed to incubate
overnight. Fluorescent Lipid Analysis of Free
Fatty Acids- Appropriate amounts of fluorescent
lipids were dried under nitrogen in sterile test
tube along with DMEM complete media. Lipids were
then resuspended by sonication, added to 35mm
dishes containing CCL39 cells at 60 percent
confluence, and allowed to incubate overnight. At
the end of incubation period, cells were rinsed
with ice-cold calcium magnesium free
phospho-buffered saline (CMF-PBS). Lipids were
extracted and separated with methanol/chloroform/0
.1M HCl (111v/v). The lower phase of the
solution was extracted and dried under nitrogen
and lipids were resuspended in 30 ?l of
chloroform/methanol (21 v/v) and applied to a
thin layer chromatography. The solvent used to
develop the TLC plate consisted of
chloroform/methanol/water (63343 v/v). The
fluorescent lipids were detected by densitometry
using UV light on the Gel Doc 1000 (Bio-Rad).