MC LACHLIN, BERNSTEIN. AND ANDERSON 
activity (e.g., lung, liver, lddney), whereas in 
tissues with greater specific activity (e.g., 
spleen, stomach, erythrocytes, lymphocytes), 
ADA is present almost solely as the small- 
molecular-weight form (15,17-19). 
Human (20-22), murine (23), and rat (24) 
ADA gene sequences have been cloned. Ex- 
pression of human ADA has been demon- 
strated in cultured mouse cells (25,26) and 
monkey cells (27) transfected with cDNA se- 
quences. Human ADA sequences which en- 
code functional protein have also been intro- 
duced into cultured mouse fibroblasts using 
a retroviral vector as a means of gene transfer 
(28). To assess the expression of human 
ADA gene sequences after introduction into 
mouse or monkey cells, human ADA has 
been separated from endogenous ADA activ- 
ity by electrophoresis in cellulose acetate gels 
(26,28), by isoelectric focusing (25), or by 
starch gel electrophoresis (27) followed by 
ADA staining in situ. Recently, a major ef- 
fort has involved the introduction of human 
ADA gene sequences into mouse and mon- 
key hematopoietic cells using retroviral me- 
diated gene transfer, which necessitated 
identifying low levels of human ADA in 
mouse or monkey peripheral blood cells. In 
the present report, we outline a procedure for 
the separation of human ADA isozymes 
from either mouse or monkey endogenous 
ADA using ion-exchange liquid chromatog- 
raphy. This method, which involves the ini- 
tial separation of ADA of different species 
from a crude cell extract and the subsequent 
concentration of partially purified protein, 
allows the resolution of even small quantities 
of human ADA activity from the back- 
ground levels of endogenous ADA enzyme. 
This technique is particularly useful when 
levels of expression of a gene introduced by a 
retroviral vector are below the limits of sensi- 
tivity for the existing methods of analysis. 
MATERIALS AND METHODS 
Chemicals. ADA, Type V from bovine 
spleen (in 50% glycerol, 50 mM potassium 
phosphate, pH 6.0) was purchased from 
Sigma Chemical Co. (St. Louis, MO). Aden- 
osine from Boehringer-Mannheim and ino- 
sine from Sigma Chemical Co. were used as 
standards in thin-layer chromatography. For 
the radiochemical ADA assay, [8- l4 C]adeno- 
sine (CFA-191) was obtained from Amer- 
sham. The ADA inhibitors 2'-deoxycofor- 
mycin (DCF) and erythro-9-(2-hydioxy-5- 
nonyl)adeninc (EHNA) were gifts from Dr. 
David Poplack (Pediatric Oncology Branch, 
NCI, Bcthesda, MD) and Dr. S. Winston 
Singleton (Burrough Wellcome Co., Re- 
search Triangle Park, NQ, respectively. 
Sample preparation. Cells in culture were 
harvested and washed twice in phosphate- 
buffered saline (PBS), and the pellet was re- 
suspended in 200-500 nl of 0.05 M KC1, 20 
mM Tris-HCl, pH 7.5. The cells were lysed 
by freeze-thawing 6 to 8 times and the sam- 
ple was spun at 12,000; in an Eppendorf 
centrifuge at 4°C for 10 min. The lysate 
could then be assayed directly for ADA activ- 
ity or fractionated by the FPLC procedure 
outlined below. 
Mononuclear peripheral blood leukocytes 
were prepared from heparinized whole blood 
using Lymphocyte Separation Medium 
(LSM) obtained from Litton Bionetics (Ken- 
sington, MD). For small volumes of blood, 4 
ml of whole blood was layered over 7 ml of 
LSM at room temperature in a 15-ml sterile 
centrifuge tube (Falcon No. 2095) and cen- 
trifuged at 3000 rpm for 8 min in a Sorvall 
RC3B centrifuge with an H-6000A rotor 
(2600;) at 25 °C. For larger samples, 15 ml of 
whole blood was layered over 25 ml of LSM 
in a 50-ml sterile centrifuge tube (Falcon No. 
2070) and spun at 3000 rpm for 10 min. The 
lymphocyte band at the plasma/LSM inter- 
face was aspirated in a small volume (2-5 
ml) and was washed with a large volume 
(35-40 ml) of PBS. The cells were pelleted at 
1600 rpm (400;) for 15 min (4°Q in a Sor- 
vall GLC-2B centrifuge with an HL-4 rotor. 
The recovered cells were resuspended in 4 ml 
of PBS and layered again over 7 ml of LSM, 
and the above procedure was repeated. After 
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Recombinant DNA Research, Volume 12 
