T-KININOGEN PRESENT IN THE LIVER OF OLD RATS IS BIOLOGICALLY-ACTIVE AND READILY FORMS COMPLEXES WITH ENDOGENOUS CYSTEINE PROTEINASES

We have previously reported an increase in T-kininogen mRNA levels in the liver of ageing Sprague-Dawley rats. T-Kininogen functions both as a precursor to the vasoactive peptide T-kinin, and as a potent and sp ecific inhibitor of cysteine proteinases. Under normal physiological c onditions, the majority of cysteine proteinases are found intracellula rly and we have shown that a significant proportion of T-kininogen als o accumulates intracellularly in the liver of old rats. Therefore, our aim was to determine whether or not this T-kininogen is biologically active as an inhibitor of cysteine proteases. Titration of whole liver extracts indicates that old rats do indeed contain a 4-fold higher le vel of cysteine proteinase inhibitory activity than younger counterpar ts. Using gel permeation chromatography in conjunction with an enzyme inhibitor assay, we show that this difference is mainly due to the pre sence of a low level of free biologically active T-kininogen. However, Western blot analysis of the gel permeation chromatography fractions demonstrate that most of the intrahepatic T-kininogen is found as enzy me-inhibitor complexes. Alkaline inactivation of the cysteine proteina se component of these complexes leads to the release of biologically c ompetent free T-kininogen. These findings are discussed with regard to the possible mechanisms responsible for the accumulation of T-kininog en within the aged rat liver. (C) 1997 Elsevier Science Ireland Ltd.