A quantitative assay for the activation of plasminogen by transformed cells in situ and by urokinase.

University of Essex, Colchester, England, United Kingdom
Biochemistry (Impact Factor: 3.19). 08/1981; 20(15):4307-14. DOI: 10.1021/bi00518a011
Source: PubMed
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    ABSTRACT: Native human plasminogen has a radius of gyration of 39 angstroms. Upon occupation of a weak lysine binding site, the radius of gyration increases to 56 angstroms, an extremely large ligand-induced conformational change. There are no intermediate conformational states between the closed and open form. The conformational chang is not accompanied by a change in secondary structure, hence the closed conformation is formed by interaction between domains that is abolished upon conversion to the open form. This reversible change in conformation, in which the shape of the protein changes from that best described by a prolate ellipsoid to a flexible structure best described by a Debye random coil, is physiologically relevant because a weak lysine binding site regulates the activation of plasminogen.
    Science 05/1990; 248(4951):69-73. DOI:10.1126/science.2108500 · 31.48 Impact Factor
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    ABSTRACT: The role of plasminogen activator (PA) in the pathogenesis of acantholysis in canine pemphigus vulgaris (PV) was evaluated using differentiated cultures of canine oral keratinocytes. Both the secreted and cell-associated PA activity in cultured canine keratinocytes were completely inhibited by specific anti-urokinase antibodies. Anti-tissue type PA antibodies did not inhibit either secreted or cell-associated PA activity. Immunoblots and fibrin zymography revealed a single 57,000 molecular weight urokinase-type PA in the conditioned media of the canine oral keratinocytes. Incubation of the differentiated cultures with PV Ig resulted in a significant increased in the levels of PA activity and both canine and human PV Ig were effective at inducing acantholysis typical of that seen in the clinical disease. The addition of urokinase inhibitor to the cultures treated with PV Ig prevented the development of acantholysis. These data strongly support the conclusion that PA is involved in acantholysis which is the cardinal feature of PV.
    American Journal Of Pathology 04/1989; 134(3):561-9. · 4.60 Impact Factor
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    ABSTRACT: The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (1P3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3 PA secretion, and cell-cell detachment in DJM-1 cells.[Ca++]i and IP3 contents were determined with or without 30-mm pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxy-methylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell- cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++] and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 µM), but not with U73343 (10 µM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 cause no effects on cell viability and IgG binding to the cell surface. These results suggest that phopholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.
    Journal of Investigative Dermatology 09/1995; 105(3):329-333. DOI:10.1111/1523-1747.ep12319948 · 6.37 Impact Factor