Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Krieger, Timothy J.; Hook, Vivian

doi:10.1016/s0021-9258(18)92986-8

Cited by 67 publications

(45 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To demonstrate the specificity of the in-gel fluorescent zymogram technique for detection of proteases, we compared the activities of trypsin and chymotrypsin, as well as a biological sample of isolated secretory vesicles from adrenal medulla (chromaffin granules) in gels copolymerized with three different peptide-MCA substrates (200 µ M each). Chromaffin granules were prepared as described previously (14,30). Different amounts of trypsin, chymotrypsin and chromaffin granules (lysed) were electrophoresed in gels containing Boc-Gln-Ala-Arg-MCA (trypsin substrate), Suc-Ala-Ala-Pro-Phe-MCA (chymotrypsin substrate) or Z-Phe-Arg-MCA (trypsin substrate).…”

Section: Methodsmentioning

confidence: 99%

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

et al. 2000

View full text Add to dashboard Cite

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.

show abstract

Section: Methodsmentioning

confidence: 99%

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

et al. 2000

View full text Add to dashboard Cite

show abstract

“…Isolation of Prohormone Thiol Protease (PTP) from Chromaffin Granules. PTP was purified from bovine chromaffin granules essentially as described previously (10). Chromaffin granules were isolated from fresh bovine adrenal medulla (obtained from Shamrock Meats, Los Angeles, CA) by discontinuous sucrose gradient centrifugation as described previously (10,19).…”

Section: Methodsmentioning

confidence: 99%

“…PTP was purified from bovine chromaffin granules essentially as described previously (10). Chromaffin granules were isolated from fresh bovine adrenal medulla (obtained from Shamrock Meats, Los Angeles, CA) by discontinuous sucrose gradient centrifugation as described previously (10,19). A soluble extract of chromaffin granules was obtained from 3 × 400 adrenal gland preparations as follows.…”

Section: Methodsmentioning

confidence: 99%

“…Our previous biochemical studies demonstrated that purified PTP consisted of a 33 kDa protein (10). However, gel filtration chromatography demonstrated PTP activity as a high molecular mass peak of 150-200 kDa.…”

mentioning

confidence: 99%

“…The yapsin-like 70 kDa aspartyl protease has been detected in chromaffin granules, but it possesses lower specific activity than PTP for cleaving the enkephalin precursor (16). Thus, the specific activity of PTP for cleaving proenkephalin (10)(11)(12)(22)(23)(24) is greater than that of the PC1 and PC2 subtilisin-like (22,(25)(26)(27) and 70 kDa aspartyl proteases (16,22).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

et al. 1999

View full text Add to dashboard Cite

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.

show abstract

Proteolysis of ProPTHrP(1–141) by “Prohormone Thiol Protease” at Multibasic Residues Generates PTHrP-Related Peptides: Implications for PTHrP Peptide Production in Lung Cancer Cells

Hook

Burton²,

Yasothornsrikul

et al. 2001

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Cited by 67 publications

References 37 publications

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

Proteolysis of ProPTHrP(1–141) by “Prohormone Thiol Protease” at Multibasic Residues Generates PTHrP-Related Peptides: Implications for PTHrP Peptide Production in Lung Cancer Cells

Contact Info

Product

Resources

About