Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a non-inhibitory mode.
OBJECTIVE
Our aim was to purify large quantities of human salivary cystatins S, SA and SN in order to determine whether these salivary cystatins have a stable interaction with cysteine proteases at a second binding site, other than the protease active site. This property may affect their availability to act as cysteine protease inhibitors within the oral environment.


METHODS
Salivary cystatins S, SA and SN were purified from human submandibular sublingual saliva to homo- geneity by column chromatography. Formation of stable complexes between the model cysteine protease papain in the absence of reductant was assessed by SDS-PAGE and probing Western blots with antibody to human salivary cystatin SN. Proteolytic activity of the complex was determined in the gel after electrophoresis.


CONCLUSIONS
Only cystatin SN (14.3 kD) was found to form a stable complex with papain (22 kD) that could be separated by SDS-PAGE producing a Coomassie stained band at (37 kD). After western transfer this same band (37 kD) cross-reacted with antibody to SN. In the presence of E64, an active site inhibitor of cysteine proteases, the same complex was formed, suggesting that SN is able to bind to papain at a site other than the active site. Activity staining of the gel confirmed that this complex (-E64) retained proteolytic activity. Such complex formation between cystatin SN and cysteine proteases in a non-inhibitory mode may reduce its availability to act as an effective cysteine protease inhibitor in the oral environment.
Baron
A
A
Department of Restorative Dentistry, University of California San Francisco, Box 0758, 707 Parnassus Avenue, San Francisco CA 94143, USA.
Barrett-Vespone
N
N
Featherstone
J
J
D015153:Blotting, Western; D002845:Chromatography; D003432:Cross-Linking Reagents; D015891:Cystatins / Q000302:isolation & purification* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D004586:Electrophoresis; D006801:Humans; D010206:Papain / Q000378:metabolism; D011485:Protein Binding; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000302:isolation & purification* / Q000378:metabolism*
C525171:CST1 protein, human; D003432:Cross-Linking Reagents; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; D010206:Papain