Regulation of serine-type exoproteinases by endogenous inhibitors present in exoantigens of the mycelial form of Paracoccidioides brasiliensis.
We have partially characterized some biochemical properties of exoproteinases secreted into culture medium by the mycelial form of Paracoccidioides brasiliensis, a dimorphic fungus that causes human disease in Latin America. Proteinase activity was analyzed in solid- and liquid-phase systems using zymography and Azocoll, respectively. Minimal or no gelatinase activity was observed by zymography in the crude filtrates among proteins with a relative mobility greater than 200 kDa. When the crude filtrate was fractionated by isoelectric focusing or ion exchange chromatography, we observed striking activation of gelatinases, both those of high apparent molecular mass and alkaline isoelectric points (pI), as well as those of lower molecular mass and acidic pI. The apparent high molecular mass gelatinases, pI 10, showed optimal activity at pH 7.0. They were totally inhibited by phenylmethylsulfonylfluoride and partially inhibited by incubation with previously neutralized fractions of pI 5.4 and 6.1. The latter inhibition could be reversed by exposure to 10% isopropanol. These results provide evidence of regulatory mechanisms controlling proteinase activity in secreted proteins. The principal mechanism appears to be the formation of reversible complexes with endogenous inhibitors.
Zambrano
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EA
Seción de Micolgía, Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Caracas. zambranoedgar@hotmail.com
Rodríguez
I
I
Mendoza
M
M
Santaella
C
C
López
M
M
Albornoz
M
M
D000946:Antigens, Fungal / Q000378:metabolism; D003470:Culture Media; D020689:Exopeptidases / Q000378:metabolism*; D005656:Fungal Proteins; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006023:Glycoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D009844:Oligosaccharides / Q000378:metabolism*; D010228:Paracoccidioides / Q000201:enzymology* / Q000254:growth & development; D011480:Protease Inhibitors / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000378:metabolism*
C103180:43 kDa protein, Paracoccidioides; D000946:Antigens, Fungal; D003470:Culture Media; D005656:Fungal Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D011480:Protease Inhibitors; D020689:Exopeptidases; D012697:Serine Endopeptidases