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 specific inhibitor of cysteine proteinases. Under normal physiological conditions, the majority of cysteine proteinases are found intracellularly and we have shown that a significant proportion of T-kininogen also 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 level of cysteine proteinase inhibitory activity than younger counterparts. Using gel permeation chromatography in conjunction with an enzyme inhibitor assay, we show that this difference is mainly due to the presence 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 enzyme-inhibitor complexes. Alkaline inactivation of the cysteine proteinase component of these complexes leads to the release of biologically competent free T-kininogen. These findings are discussed with regard to the possible mechanisms responsible for the accumulation of T-kininogen within the aged rat liver.
Keppler
D
D
Experimental Pathology Unit, Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland.
Sordat
B
B
Sierra
F
F
D000375:Aging / Q000378:metabolism / Q000502:physiology*; D000468:Alkalies; D000818:Animals; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000502:physiology*; D007704:Kininogens / Q000737:chemistry / Q000502:physiology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D000468:Alkalies; D015853:Cysteine Proteinase Inhibitors; D007704:Kininogens; D003546:Cysteine Endopeptidases