The assay of glandular kallikrein and prekallikrein in human mixed saliva.
The measurement of glandular kallikrein in biological fluids most often utilizes a synthetic substrate, H-D-valylleucylarginine-p-nitroanilide (S-2266), which assesses amidase activity. Although this substrate has reasonable specificity for glandular kallikrein, other tryptic-like proteases found in mixed saliva may also cause hydrolysis. The primary purpose of this study was to assess the accuracy of the use of this substrate for the measurement of glandular kallikrein in human mixed saliva. An additional objective was to determine the presence of prekallikrein in mixed saliva. The addition of soybean trypsin inhibitor (SBTI), which inhibits other tryptic-like enzymes but not glandular kallikrein, resulted in an approx. 30 per cent decrease in the hydrolysis of S-2266 by centrifuged mixed human saliva. A correlation of 0.918 was obtained between the biological assays for kinin release and amidase activity in 19 subject samples. Amidase activity increased following treatment of saliva with trypsin, indicating the presence of prekallikrein in human mixed saliva. It is concluded that S-2266 is an accurate substrate for the assay of glandular kallikrein in human mixed saliva; that the inclusion of SBTI in the assay mixture is needed to inhibit non-kallikrein proteases that may also hydrolyse the synthetic substrate; and that prekallikrein is present in mixed saliva. Thus any future studies of changes in the level of kallikrein in saliva may wish to consider the presence of both active and total levels of glandular kallikrein.
Jenzano
J W
JW
Dental Research Center, School of Dentistry, University of North Carolina, Chapel Hill 27599.
Coffey
J C
JC
Heizer
W D
WD
Lundblad
R L
RL
Scicli
A G
AG
D000581:Amidohydrolases / Q000032:analysis; D006801:Humans; D007610:Kallikreins / Q000032:analysis*; D009842:Oligopeptides; D011288:Prekallikrein / Q000032:analysis*; D012463:Saliva / Q000187:drug effects / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology; D014361:Trypsin Inhibitors
D009842:Oligopeptides; D014361:Trypsin Inhibitors; C031006:Val-Leu-Arg-p-nitroanilide; D011288:Prekallikrein; D007610:Kallikreins; D014357:Trypsin; D000581:Amidohydrolases