Abstract
The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.
Highlights
simian virus 40-transformed 3T3 (SV3T3) cells than by the 3T3 cells
We conclude that the elevated capacity of SV3T3 cells t o degrade hyaluronate maybe partially responsible fotrheir lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells
We have begun to investigate whether these findings are a reflection of different rates of degradation of hyaluronate in the two cell lines, such that 3T3 cells, which have relatively high endogenouslevels of hyaluronate and which bindsmaller amounts of exogenous hyaluronate, degrade hyaluronate to a lesser degree than do their transformed counterparts
Summary
Cell Lines and Culture Procedures-The 3T3 cell line was obtained from Dr J. Samples (5200 pronase was added (final concentration of 3 mg/ml) and the incubap l ) were incubated at 37 "C for 16 h with hyaluronate substrate Binding and Degradatioonf Radiolabeled Hyaluronate"SV3T3 and the NaCl concentration of the buffer (11, 12) prevented exoglycosidaseaction from interfering with the measurement of hyaluronidase activity In contrast to these findings, cultures of both cell types were found to contain the exoglycosidasesP-N-acetylglucosand3T3 cells were grown to confluency in 100-mm tissue culture aminidase and P-glucuronidase (Table I).The former enzyme dishes and refed with serum-containing medium prior to experimental manipulation. All plates were given radiolabeled hyaluronate at a final concentration of p g / d a t was present in the cell layer and was secreted into the culture medium, whereas the latter was detected only in the cell layer. Unlabeled carrier hyaluronate was added to a final concentration of 1.3-1.5 mg/ml of culture medium
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