Download Membrane peptidase activity of a human endothelial cell line (EA.hy

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Biochemical Society Transactions ( 1 994) 22
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Memhrnne peplldsse ncllvlty o l n human endotbelbl cell Une CA.hy 926)
Table I
KAlIIIiKINI~J GREENHOUGH. BRENDAN J WALKDEN. LEONARD J
MIJKPIIY, IIALlL I: MCLAREN. KAY BARNES and ANTHONY J
'IUKNIiK
EA.hy 926 cell membranes (approx. I mg/ml) were incubated with detergent
at 4OC for 2 h and the solubilised activity determined after centrifugation at
100 Ooog for 1.5 h. E-24.1I activity was assayed by a hplc method [lo] and
ECE activity assayed with Rig ET-I (I pM) as substrate and lhe ET-I
produced determined using an RIA [II] specific for the C-terminal ET-I (162 I)sequence.
Ikparunent of Biochemistry and Molecular Diology. University of Leeds.
I.eeds. LS2 9 J l U.K.
The 1iA.hy 926 cell line is a permanent human endothelial cell line
originating from the fusion of primary human umbilical vein endothelial cells
and cells from the human lung carcinoma cell line A549 [ 11. The EAhy 926
cell line shows sustained expression of many differentiated functions of the
vascular endothelium 111. I t has also been reponed to express mRNA for
endothelin-I(ET-l) and lo secrete both ET-I and its precursor Big ET-I 121.
The processing of Big ET-I to ET-I occurs by a putative endolhelinconvening enzyme 131, which exhibits phosphoramidon sensitivity and bears
some similarity to endopeptidase-24.1 I (E-24.11. EC 3.4.24.11) 141. A
putative endothelin convening enzyme (ECE) activity processing Big ET-I to
13'- I has been reponed in EAhy 926 cells [S]. Here we have investigated lhe
profile of membrane peplidases expressed by the cell line and determined the
nature of an endothelin-convercing enzyme activity.
EA.hy Y26 cells were cultured in Dulbecco's modified Eagle medium
supplemented with HAT (100 pM hypoxanthine, 0.4 pM aminopterin. 16 pM
thymidine). 40 mM glutamine. 10% foetal calf sem. 100 IU/ml penicillin,
and 100 pg/ml streptomycin. at 37"C in 5% CO2 in air.
To prepare
membranes cell monolayers were rinsed well then scaped into 50 mM TrisHCI. IOOmM NaCI. 18 mM CaC12. pH 7.4 and bomongenised by N2
cavitation (800 psi at
for 10 min) in a PARR cell disruption bomb.
Homogenates were centrifuged (IOOOg. 10 min) to remove nuclei and cell
debris and the supemalants centrifuged again (100 Ooog. 90 min) 10 yield
membrane pellets which were resuspended by hand homogenisalion in 50 mM
Tris-HCI , IOOmM NaCI. pH 7.4.
lmmunostaining of EAhy 926 cells, cultured 2-3 days on covenlips. with
secondary fluorescent antibodies 161 revealed limited expression of cellsurface peptrdases. Staining with a monoclonal antibody (Saocec. MCA 659)
lo human aminopeptrdase N resulted in bright fluorrscence of lhe
plasmalemma typical of a cell-surface pepcidase. Faint flwreswnce was also
seen with RP161. a polyclonal antibody lo porcine endopepcldase-24.I I . Cells
incubated with antibodies D DPPIV. ACE or with pre-immune serum did IK)t
exhibit fluorescence. Tbts limited cell surface peplidase expression was also
reflected in lhe enzyme a c t i v i h of membranes prepared from the cultured
EA.hy 926 cells. Endopepcidase-24.1I (20.88 nmol/min/mg), aminopeptidase
N (60.33 nmol/min/mg) and an endothelin converting enzyme activity (55.83
pmol/min/mg) were dewled in EA.hy 926 cell membranes. ACE, DPPIV.
AP-A. AP-P and alkaline phosphalase, however. were no( delecled. The l?.lE
activity of the cell membranes was distinct from E-24. I1 membrane activity,
which also hydrolyses Big ET-Iand ET-I 171, in bat it was phospboramidonsensitive but thiorphan-insensitive.
To determine h e nature of the ECE activity EAhy 926 cell membranes were
subjected to phase separation lhrough Triton XI14 IS]. Bolb E-24.11 and
ECE were panitioned predominanlly in lhe detergent phase (93.4% and 79.1%
respectively).
In comparison LDH. a cytosolic enzyme. pUUlioncd
predominantly into lhe aqucous phame. Treament of EA.hy 926 cell
membranes with eilher octyl glucosidz Nonidet P-40 or Triton XI00 at 4"C
resulted in equal soluhilisation of ECE by all the detergents. E-24.11.a
Vansmemhrane anchored protein. exhibited a similar profile (Table I).
Incubation of EA.hy 926 cell membranes with bacterial PI-PLC a l 3 W failed
to solubilise either E-24. I I or ECE.
In conclusion, EA.by 926 cells exhibit limited e x p s i o n of cell-surface
pepcidases but do express an enzyme activity distinct from E-24.11 lhat
hydrolyses Big ET-I to its active form ET-I. This ECE activity is
phosphoramidon sensitive but in convar~ lo E-24.11 is insensitive to
hiorphan. The ECE activity has been eslablisbcd as an integral membrane
protein by Triton X-I14 phase sepantion and from the pattern of detergent
solubilisation and resistance to release by bacterial PI-PLC the ECE activity
appears to be atlached to the membrane by a bybopbobs lfansmembraoe
domain 191.
Abbreviations used:
Big ET-I, big endolhelin-I; ET-I. endohelin-I; ECE, endolhelin convening
enzyme; E-24.11. endopcptrdase-24.Il; DPPIV. dipepcidyl peptidase IV;
ACE, angiotensin convening enzyme; AP-A. aminopepcidase A; AP-P.
aminopeptrdase P; LDH, lactate dehydrogenase; PI-PLC, pbosphatidylinosild
specific phospholipase C.
Detergent
Final
concentration
(mM)
Solubilised Activity
(% of total membrane activity)
E-24. I I
None
Oktyl Glucoside
Nonidet P-40
Triton X-100
60
6.1
6.0
0
91.2
94.1
87.0
ECE
0
53.6
75.3
72.8
We thank Ih C J kidgel1 for the EA.hy 926 cell line and Dr K Corder for
donation of the antibody to C-terminal ET-I (16-21) sequence. The work was
supported by the British Heart Foundation.
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