Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
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PMID:Expression of rat kallikrein and epithelial polarity in transfected Madin-Darby canine kidney cells. 749 Jan 45

Pefabloc and Trasylol are serine protease inhibitors that have been used during islet isolation to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) and improve islet yields. Using in vitro studies, we evaluated the effects of Pefabloc and Trasylol on the activities of these proteases and the effect of Pefabloc on islet function. In addition, it has been reported that Protector Solution (PS) enhances the efficiency of Pefabloc. Thus, we evaluated the efficacy of Pefabloc in the presence or absence of PS. EnzCheck protease assay was used to measure the activities of trypsin, chymotrypsin, elastase, liberase, and thermolysin in the presence or absence of 0.4 mmol/L Pefabloc (with or without PS) or 0.43 micromol/L Trasylol. We also tested switch samples containing the highest concentration of enzymes. Pefabloc significantly inhibited trypsin, chymotrypsin, elastase, and switch, but not liberase or thermolysin. Trasylol significantly inhibited all enzymes except for elastase and switch sample. Unexpectedly, the potency of Pefabloc was abrogated when diluted first in PS. Insulin release was diminished when islets were incubated or perifused with Pefabloc. In conclusion, Pefabloc when added appropriately significantly blocked in vitro protease activity. Unfortunately, Pefabloc also decreased islet insulin secretion, making it unsuitable for islet isolation. Trasylol cannot be used with collagenase because it impaired both liberase and thermolysin.
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PMID:Endogenous pancreatic protease activity and methods for impeding their function. 1837 65

Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.
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PMID:Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases. 2109 14