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Birckbichler & Patterson: Cellular Characteristics
355
TGase activity." The differential in activity was independent of several homogen-
ization procedures, not a result of incomplete cell rupture, and not the result of
endogenous activators or inhibitors of TGase in the cells. Similarly, the possibility
that the results were due to plasma TGase contamination of the preparations was
minimal. Normal and transformed preparations exhibited similar Michaelis
values for the amine donor putrescine, suggesting that the affinity of the enzyme
for this substrate was unaffected by transformation." Furthermore, these findings
suggested that the reduction in activity was independent of the transforming agent,
and there were differential levels of TGase-catalyzed cross-links in normal and
transformed cells.
TABLE 1
TRANSGLUTAMINASE ACTIVITY IN NORMAL AND TRANSFORMED CELLS
Specific Activity*
Normal
Cell/Tissue
Source
(U/mg of protein)
Transformed
Liver
rat
8.5 + 0.4+ (8)1
Novikoff hepatoma
rat
2.0 + 0.1 (6)
4.3
Primary hepatomas
rat
5.4 + 0.5 (10)
1.6
WI-38
human
7.0 + 1.0 (7)
WI-38 VA13A
human
0.12 + 0.02 (7)
58.3
WI-38 VA13-2RA
human
0.04
175.
BHK-21/C13
hamster
1.1 + 0.2 (3)
PyBHK-21/C13
hamster
0.03 + 0.02 (3)
36.7
C3H/10T1/2CL8
mouse
0.28 + 0.10 (3)
C3H/10T1/2CL8MCATCL15
mouse
0.03 + 0.01 (3)
9.3
*Homogenate activity except for C3H system, which is the 25,000 g supernatant.
+ Means + SE.
Numbers of preparations assayed.
$Induced with 3'-methyl-4-dimethylaminoazobenzene.
E-(7-GLUTAMYL)LYSINE IN NORMAL AND TRANSFORMED CELLS
To complement the enzyme studies, quantitativenzyme product, c-(y-
glutamyl)lysine, was performed. The isopeptide bond is resistant to proteolytic
attack, so that after exhaustive enzyme digestion by pronase and leucine amino-
peptidase, <-(-glutamyl)lysine can be identified by amino acid chromatography.
In most cases, more than 80% of the protein is converted to free amino acids and
isopeptide during enzymatic digestion. Analysis of pronase-digested total cellular
protein revealed that normal WI-38 cultures contained 40-100 times more e-(y-
glutamyl)lysine bonds per cell than did WI-38 VA13A cultures (TABLE 2). These
values agree with the enzymatic differential shown in TABLE 1.
IMMUNOFLUORESCENT STUDIES
A differential between WI-38 and WI-38 VA13A cultures was also demon-
strated when cells were stained by indirect immunofluorescence with rabbit anti-
TGase antiserum. Sparse WI-38 cultures produced a weak intracellular fluor-
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