Immunoaffinity Chromatographic Purification of Chicken ectoATP Diphosphohydrolase R. STROBEL AND M. ROSENBERG Genetics and Cell Biology University of Minnesota St. Paul, Minnesota 55108 The surfaces of all cells contain a variety of enzyme activities. Ectoenzymes are integral membrane proteins whose catalytic activities are directed externally. One common ectoenzyme is ectoATPase, found on most nucleated cells. It is Ca2+ or Mg2+ dependent, has low affinity (K,,, around 0.5 mM),and also hydrolyzes ADP (therefore it is an ATP diphosphohydrolase). It is inhibited by azide and fluoride, but not by classical ATPase inhibitors.

210

116

-

97-

43

I.

9

MW standards

Oviduct

Liver

FIGURE 1. Silver-stained SDS-PAGE gel of purified oviduct and liver ATPases.

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ANNALS NEW YORK ACADEMY OF SCIENCES

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Our initial goal was to purify the ectoATPase from chicken oviductal fluids, where the enzyme is associated with a population of 400 nm microvesicles shed from the oviductal epithelial cells. The enzyme was partially purified using gel filtration, solubilization in NP-40, DEAE and lentil lectin chromatography, and preparative native gel electrophoresis. SDS-PAGE of the most purified fraction revealed several bands. The next phase of the project involved generation of monoclonal antibodies to this partially purified oviduct ectoATPase. Mice were immunized with homogenized MONOCLONAL ANTIBODY SCREENING ASSAY 1. 96-WELL PVC (COSTAR) MICROTITER PLATES

2. RABBIT-ANTI-MOUSE IgG (R LABORATORY

3. 200

A

01

M) PREPARED IN

(0.1 MG/ML, R a M) TO WELLS, 4" C, 12 HRS

4. I%BSA BLOCK

5. MONOCLONAL ANTIBODY SUPERNATANTS, 37" C, 4 HRS 6. ATPDAsE, 200

A,

37" C, 4 HRS, WITH AGITATION

7. 200 x, 4 mM ATP, 37" C, 12 HRS 8. SUPERNATANT TESTED FOR FREE PHOSPHATE

I

ATP

AMP

+ Pi + PiI

I

Monoclonal Mouse Anti-ATPDase

4

!

Rabbit Anti-Mouse IgG

FIGURE 2. Monoclonal antibody screening assay.

gel bands from preparative native gels in which the enzyme was located using an ATPase-specificzymogram. Anti-ATPase specifichybridomas were selected using an ATPase-specific enzyme activity capture assay. Briefly, PVC microtiter plates were sequentially coated with rabbit anti-mouse IgG, hybridoma supernatant, and solubilized ATPase. An ATPase assay was then performed in the wells, and the phosphate generated was visualized using a colorimetric assay. Six stable, twice-cloned hybridomas to the oviductal ectoATPase were generated. All six antibodies were bound to hydrazide-activated agarose gels and screened for usefulness in affinity chromatography of the oviductal ectoATPase. One (desig-

STROBEL & ROSENBERG: ectoATP DIPHOSPHOHYDROUSE

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nated MC22) was found to be particularly useful. With this antibody, the oviductal ectoATPase was purified to homogeneity. It consists of a single band of 80K. It has a specific activity of 806 unitslmg protein. We also purified the chicken liver ectoATPase using a similar protocol. Chicken liver is homogenized and a crude membrane fraction isolated using differential centrifugation. ATPase was solubilized using NP-40 and partially purified by DEAE and ConA chromatography. The partially purified enzyme is then purified to homogeneity using affinity chromatography on the monoclonal antibody column. The purified liver enzyme consists of a single band of 90K. It has a specific activity of 1,246 units/mg protein. Studies are currently in progress to determine the cross-reactivity of the monoclonal antibodies in other species. Preliminary results demonstrated cross-reactivity with rat liver ectoATPase. The enzyme is being sequenced to obtain sufficient information to develop oligonucleotide probes useful for cloning the gene. The monoclonal antibodies are being used to study the tissue distribution of the enzyme. REFERENCE

1. STROBEL,R. S., D. S. Lru & M. D. ROSENBERG. 1991. Purification of ecto-nucleotide diphospholase from reproductive system and liver. J. Cell Biol. 115: 59a.