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s
52K =-._
SOl(
p m p 234
IgG
Fig. 2. SR-D virus was disrupted
with (2%) NP-40, centrifuged
at 45.000 rpm for 180 min
at 4 oe and separated into supernatant
(s) and pellet (p). The
pellet was used for membrane
isolation (m) according to a published
procedure (Van de Ven
et al. 1978). Identical aliquots
of s, p and m were incubated for
endogenous
phosphorylation
and IgG phosphorylation
(Owada and Moelling 1980).
The purified enzyme from the
phosphocellulose colum was incubated
for endogenous phosphorylation
under various eonditions
to demonstrate its proteolytic
cleavage. Incubation was in 1: 60 min, ooe, pH 6.8; 2: 30 min, 20 o e, pH 6.8; 3. 2 h, ooe, pH 6.8; and
4: 30 min, 20 o e, pH 8.2
Table 1. SR-D without and with disruption (2 % NP-40) was used for IgG phosphorylation, for precipitation
of 3H-glueosamine radioactivity of the gp85 by antibodies, for surface iodination by means of lactoperoxidase
(NEN, Radioeh.), and for endogenous phosphorylation. Trypsin treatment (10 !!g/ml) was for 30 min at room
temperature (nt: not tested)
Trypsin
treatment
IgG-phosphorylation
(epmX 10 3 )
H3-gluco
samine
gp85 (epm)
125J_pp60src
125J_gp85 Endogenous phosphorylation
(cpm)
32p-60K 32P-50/52K P 19
(epm) (%)
Intaet
Virus +
Disrupted
Virus +
52 1360
58 603
512 964
564 537
0 1064 0 0 100
0 532 0 0 100
169 809 77 610 100
146 462 72 680 2
Isolated
Membranes +
nt
nt
nt
nt
nt nt 452 41 nt
nt nt 39 398 nt
viral envelope
or
(eil membrane
gp37
~'J':
S'SO
D
gp85
Fig. 3. Sehematic presentation of the loeation of
pp60 src in an avian RNA tumor virus particle. gp,
glycoprotein
407