Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells.

Abstract

The intracellular distribution of a specific protease, plasminogen activator (P,~/), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution.A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, and endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na +,K+-ATPase, 5'-nucleotidase, and [aH]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like" elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.Enhanced proteolytic activity has been correlated with the malignant state in vivo where it has been demonstrated that increased levels of proteolytic enzymes are found in the tissues as well as the plasma and other fluids of many species of animals exhibiting a variety of malignant tumors (6,20, 472

Immunohistochemical localisation of tissue plasminogen activator in human brain tumours

“…The pattern of cellular distribution also differs, with granular/punctuate positivity in endothelium and uniform surface reactivity in the few parenchymal cells that reacted. These observations are consistent with some of the activity being lysosomal in endothelial cells as has been found in other species and tissues (Ali & Lack, 1965), and the finding that in vitro tumour cell PA is membrane associated (Quigley et al, 1976).…”

Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

“…In a number of published studies of cells other than neutrophils, PA was found in the plasma membrane or plasma membrane-like structures (8,11,12), but in view of our findings, which clearly show that other cell proteases may have a crucial effect on the quantity and the distribution of PA, these results should be reexamined .…”

Synthesis of fibrinolytic activator and inhibitor by endothelial cells

“…In spite of these observations, it has been difficult to demonstrate that Pmg As are actually synthesized in this tissue (9). The (27,28), possibly with the plasma membrane (11). Intracellular Ping A was found to exist in a latent form that could be revealed by brief, controlled acidification.…”