Tài liệu Báo cáo Y học: Purification, characterization, cloning, and expression of the chicken liver

Purification, characterization, cloning, and expression of the chicken

liver ecto-ATP-diphosphohydrolase

Aileen F. Knowles1

, Agnes K. Nagy2

, Randy S. Strobel3 and Mae Wu-Weis1

1

Department of Chemistry, San Diego State University, San Diego, CA, USA; 2

West Los Angeles Veterans Affairs Medical Center,

Los Angeles, CA, USA; 3

Department of Natural Sciences, Metropolitan State University, St Paul, MN, USA

We previously demonstrated that the major ecto-nucleoside

triphosphate phosphohydrolase in the chicken liver

membranes is an ecto-ATP-diphosphohydrolase (ecto￾ATPDase) [Caldwell, C., Davis, M.D. & Knowles, A.F.

(1999) Arch. Biochem. Biophys. 362, 46–58]. Enzymatic

properties of the liver membrane ecto-ATPDase are similar

to those of the chicken oviduct ecto-ATPDase that we have

previously purified and cloned. Using antibody developed

against the latter, we have purified the chicken liver

ecto-ATPDase to homogeneity. The purified enzyme is a

glycoprotein with a molecular mass of 85 kDa and a specific

activity of
1000 UÆmg protein)1

. Although slightly larger

than the 80-kDa oviduct enzyme, the two ecto-ATPDases

are nearly identical with respect to their enzymatic properties

and mass of the deglycosylated proteins. The primary

sequence of the liver ecto-ATPDase deduced from its cDNA

obtained by RT-PCR cloning also shows only minor

differences from that of the oviduct ecto-ATPDase.

Immunochemical staining demonstrates the distribution of

the ecto-ATPDase in the bile canaliculi of the chicken liver.

HeLa cells transfected with the chicken liver ecto-ATPDase

cDNA express an ecto-nucleotidase activity with character￾istics similar to the enzyme in its native membranes, most

significant of these is stimulation of the ATPDase activity by

detergents, which inhibits other members of the ecto￾nucleoside triphosphate diphosphohydrolase (E-NTPDase)

family. The stimulation of the expressed liver ecto-ATPDase

by detergents indicates that this property is intrinsic to

the enzyme protein, and cannot be attributed to the lipid

environment of the native membranes. The molecular

identification and expression of a liver ecto-ATPDase,

reported here for the first time, will facilitate future

investigations into the differences between structure and

function of the different E-NTPDases, existence of liver

ecto-ATPDase isoforms in different species, its alteration in

pathogenic conditions, and its physiological function.

Keywords: ecto-ATP-diphosphohydrolase; chicken liver;

E-NTPDase; expression; immunoaffinity purification.

E(cto)-ATPases (E-ATPases; also known as E-NTPDases)

(EC 3.6.1.5) are ubiquitous cell surface glycoproteins that

hydrolyze nucleoside triphosphates. Some will also hydro￾lyze nucleoside diphosphates. Their physiological substrates

are probably the ligands of purinergic receptors, e.g.

extracellular ATP, ADP, and UTP [1]. They may also play

a role in regulating substrate concentration of ecto-protein

kinases [2]. A substantial literature on the characterization

of the E-ATPases in intact cells and plasma membrane

preparations has accumulated since the 1970s (reviewed in

[3]). Because of their low abundance and the lability of some

E-ATPases to detergents, only the E-ATPases of rabbit

muscle transverse tubules [4], chicken gizzard [5], human

placenta [6], and chicken oviduct [7] have been purified to

homogeneity. On the other hand, the cDNA sequences of

more than two dozen related E-ATPases and soluble E-type

ATPases have been reported, establishing an E-ATPase

gene family [8]. The cDNAs of membrane-bound

E-ATPases encode proteins of
500 amino acids. The

bulk of the E-ATPase protein is extracellular with two

transmembranous domains near the N- and C-termini.

Variable numbers of potential N-glycosylation sites and

protein kinase consensus motifs occur in the sequences.

More importantly, all contain five highly conserved apyrase

consensus regions [9,10] and 10 conserved cysteine residues,

the latter are probably involved in disulfide bond formation.

The E-ATPases can be divided into two groups based

on their substrate selectivity and inhibition by azide.

The ecto-ATP-diphosphohydrolases (ecto-ATPDases or

ecto-apyrases) hydrolyze NDPs as well as NTPs and are

inhibited by high concentrations of azide, whereas the ecto￾ATPases show little activity toward NDPs and are not

inhibited by azide. The ecto-ATPDases are comprised of

different isoforms. The majority of the ecto-ATPDases

that have been cloned are closely related to CD39, a cell

surface antigen that is expressed on activated lymphocytes

[11,12]. CD39s from several species have 60–90% identity

in their primary sequences [12–15]. Biochemical and

Correspondence to A. F. Knowles, Department of Chemistry,

San Diego State University, San Diego, CA 92182-1030, USA.

Fax: + 1619 594 4634, Tel.: + 1619 594 2065,

E-mail: [email protected]

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium.

Definitions: E-ATPases are a family of cell surface (ecto) ATPases that

hydrolyze extracellular ATP; they are also known as the E-NTPDases.

Ecto-ATP-diphosphohydrolase (ecto-ATPDases) and ecto-ATPases

are two different subfamilies of the E-ATPases. E-type ATPases are

ATPases that have similar enzymatic characteristics and sequence

homology to the E-ATPases, however, they are not membrane

proteins.

(Received 17 December 2001, revised 18 March 2002,

accepted 22 March 2002)

Eur. J. Biochem. 269, 2373–2382 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02898.x