Purification of human kallikrein 6 from biological fluids and identification of its complex with alpha(1)-antichymotrypsin.
BACKGROUND
Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized.


METHODS
hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum.


RESULTS
N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of approximately 25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that alpha(1)-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum.


CONCLUSIONS
hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with alpha(1)-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.
Hutchinson
Shirley
S
Department of Pathology, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
Luo
Liu-Ying
LY
Yousef
George M
GM
Soosaipillai
Antoninus
A
Diamandis
Eleftherios P
EP
D015415:Biomarkers / Q000737:chemistry; D015153:Blotting, Western; D001826:Body Fluids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015200:Fluoroimmunoassay; D006801:Humans; D007610:Kallikreins / Q000737:chemistry* / Q000302:isolation & purification*; D010051:Ovarian Neoplasms / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry*
D015415:Biomarkers; D011480:Protease Inhibitors; D011498:Protein Precursors; D000514:alpha 1-Antichymotrypsin; C103551:KLK6 protein, human; D007610:Kallikreins