Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells.
Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5 x 10(6) cells in 24 hours cleaved 46 nmol/min D-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D007150:Immunohistochemistry; D007610:Kallikreins / Q000235:genetics* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013363:Submandibular Gland / Q000201:enzymology; D013997:Time Factors; D014162:Transfection / Q000235:genetics
D003470:Culture Media; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D007610:Kallikreins