MC LACHLIN, BERNSTEIN. AND ANDERSON 
tions for separating ADA of different species. 
It was determined that the salt concentration 
and pH of the column fractions containing 
ADA did not interfere with enzyme activity 
in subsequent assays. In all cases, clarified 
whole cell extracts were fractionated on an 
Mono Q column (HR 5/5), and fractions 
were combined in pairs and concentrated ap- 
proximately 20-fold by ultrafiltration (see 
Material and Methods). ADA activity in col- 
umn fractions was demonstrated by starch 
gel electrophoresis with in situ histochemical 
staining and/or by measuring the conversion 
of radioactively labeled adenosine to inosine. 
Lysates of human peripheral blood mono- 
nuclear cells and murine NTH 3T3 cells were 
fractionated on separate Mono Q columns, 
and the combined fractions were assayed for 
ADA by conversion of [ |4 C]adenosine (Fig. 
1). When a lysate of human peripheral blood 
mononuclear cells was fractionated on a 
Mono Q column and assayed for ADA activ- 
ity, one major and one minor peak of en- 
zyme activity were detected (open circles). 
The majority of human ADA did not bind to 
the column and eluted in fractions 2-4 while 
a small peak of activity was detected in frac- 
tions 19 and 20. It was not possible to find 
conditions on either Mono Q (a strong anion 
exchanger) or Mono S (a strong cation ex- 
changer) columns under which human ADA 
would bind. When the same ADA-contain- 
ing fractions were analyzed by starch gel elec- 
trophoresis (data not shown), the major peak 
of activity in fractions 2-4 had an isozyme 
pattern characteristic of red cell ADA while 
the minor peak of activity had less electro- 
phoretic mobility, similar to that previously 
described for tissue ADA (13,14). The ob- 
served ADA activity from both peaks of the 
human sample was completely inhibited by 
both DCF and EHNA at concentrations of 
10 and 100 mm, respectively. Both DCF and 
EHNA are inhibitors of adenosine deamin- 
ase (3 1 ). A single peak of murine ADA activ- 
ity was- identified (closed circles). Lower 
levels of ADA activity are cleared detectable 
in fractions 15 and 16 and 23 and 24 by the 
radioactive assay, whereas in the same sam- 
ple analyzed by starch gel electrophoresis de- 
monstrable ADA activity was found only in 
fractions 17 and 18 and low levels of staining 
in fractions 19 and 20, indicating the lower 
sensitivity of the start gel method of ADA 
analysis. The effect of loading a larger 
amount of protein from 3T3 cells is also il- 
lustrated. There was no evidence of murine 
ADA eluting from the column in the frac- 
tions containing the majority of human 
ADA (3 and 4). 
Similar results were obtained when a lysate 
from 10* mononuclear cells from macaque 
bone marrow was fractionated on a Mono Q 
column. A broad peak of endogenous mon- 
key ADA was identified, with the majority of 
the activity eluting in fractions 19 and 20 as 
assayed by starch gel electrophoresis. When 
the assay was performed by measuring the 
conversion of [ |4 C]adenosine, there was no 
detectable ADA activity in fractions 2-6 
where human ADA elutes (data not shown). 
We next demonstrated that NIH 3T3 cells, 
when infected with a Moloney-based retro- 
viral vector containing the human ADA 
cDNA called SAX (32), will express human 
ADA which can be separated from the en- 
dogenous mouse ADA by FPLC Mono Q 
fractionation. A whole-cell extract of SAX- 
infected 3T3 cells was fractionated on the 
column and then assayed for ADA activity 
by starch gel electrophoresis. The protein 
profile of the FPLC column is shown in Fig. 
2A. Human ADA isozymes were demon- 
strated in fractions 3-6, and the endogenous 
mouse activity was present in fractions 
17-20 when concentrated column fractions 
were analyzed by starch gel electrophoresis 
(Fig. 3). These results show that the human 
ADA sequences introduced by a retroviral 
vector were expressed in mouse cells and 
eluted from the FPLC column in the same 
region that the majority of human ADA in 
control human cells is detected. It is interest- 
ing to note that the three characteristic iso- 
zymes of human ADA are produced in a 
mouse cell line, suggesting appropriate post- 
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Recombinant DNA Research. Volume 12 
