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Birckbichler & Patterson: Cellular Characteristics
359
were effective in increasing TGase activity, with trypsin producing the largest
stimulation. Note that thrombin was ineffective in activating TGase. Further evi-
dence for a nonmitogenic role for trypsin activation is that high TGase activity
appears to be contrary to the proliferative state in this system (FIGURE 2 &
TABLE 1). Rather, the activation seemed to be related to some other consequence
of trypsin administration.
TRANSGLUTAMINASE AND FIBRONECTIN
The rapidity with which trypsin activated TGase suggested that hydrolytic
cleavage of a cell surface component might be involved, with cleavage of this com-
ponent apparently leading to solubilization of TGase activity. Similarly, the dif-
ferential effects of trypsin and pronase as compared to that of thrombin on ac-
tivating TGase parallels their action in cleaving a cell surface glycoprotein found in
several fibroblast cells. This glycoprotein has received many names, including
TABLE 3
TRANSGLUTAMINASE ACTIVITY AFTER MITOGEN TREATMENT
Treated Activity
Treatment*
Concentration
Untreated Activity
Medium changet
0.9, 0.9
CaCl2
1.8-5 mM
1.1 # 0.2 (3)
Trypsin
Sug/ml
2.7 + 0.3 (5)
Pronases
1.7, 1.8
Thrombin
5 ug/ml
5 ug/ml
1.1, 1.3
*Cultures were washed (except those that received the medium change) and incubated
with the appropriate reagent in Earle's solution for 1 hr at 37 C prior to assay.
+Modified Mccoy's medium Sa (2) plus 10% fetal bovine serum (Reheis Chemical Cor-
poration).
* Worthington Biochemical Corporation.
$Calbiochem.
"Parke-Davis, topical.
fibronectin, which will be used in this discussion. Most of these surface proteins
are thought to be the same protein and cross-react immunologically with the serum
protein, cold-insoluble globulin." These proteins are apparently transformation
sensitive, since they are lost from the cell surface after transformation.20. Trypsin
and pronase catalyze removal of fibronectin from fibroblasts, whereas thrombin
fails to do so under similar conditions." To determine whether a relationship be-
tween TGase and fibronectin exists, a comparative study was undertaken.
Initially, surface fibronectin in WI-38 cells was labeled by the lactoperoxidase-
catalyzed iodination procedure with 121." Addition of trypsin to these labeled
cells resulted in an enzyme-dependent loss of 60-75% of the radioactive label
within 30 min. While the I studies provided circumstantial evidence of fibronec-
tin involvement, we desired to confirm these findings by another method.
WI-38 and WI-38 VA13A cells demonstrated a differential in fluorescence
when stained by indirect immunofluorescence with goat antifibronectin antiserum
(FIGURE 3). Preconfluent, growing WI-38 cells showed essentially an intracellular
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