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Studies on rat renal cortical cell kallikrein. I. Separation and measurement.
Metadata
JournalBiochim. Biophys. ActaNot FoundDate
1975-Dec-05
Publication Type
Research Support, U.S. Gov't, P.H.S.
Journal Article
Volume
1975-Dec-05 / 411 : 305-15
Author
Kaizu T , Margolius HS
DoiPMIDMESH
Aldosterone
Animals
Biological Assay
Hydrogen-Ion Concentration
Immunodiffusion
Kallikreins
Kidney
Kidney Cortex
Kinetics
Male
Organ Specificity
Rats
Spironolactone
Abstract
A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.
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