scholarly journals Limited proteolysis by macrophage elastase inactivates human alpha 1-proteinase inhibitor.

1980 ◽  
Vol 152 (6) ◽  
pp. 1563-1570 ◽  
Author(s):  
M J Banda ◽  
E J Clark ◽  
Z Werb
Keyword(s):  
Molecular Weight ◽  
Proteinase Inhibitor ◽  
Limited Proteolysis ◽  
Serine Proteinase ◽  
Apparent Molecular Weight ◽  
Peritoneal Macrophages ◽  
Granulocyte Elastase ◽  
Inhibitor Complex ◽  
Mouse Peritoneal Macrophages

Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit human granulocyte elastase, a serine proteinase implicated in the pathogenesis of emphysema. Macrophage elastase apparently does not degrade human granulocyte elastase-alpha 1-proteinase inhibitor complexes or release active granulocyte elastase from these complexes. The ability of macrophage elastase to degrade alpha 1-proteinase inhibitor is inhibited by EDTA and alpha 2-macroglobulin.

PRODUCTS OF LIMITED PROTEOLYSIS OF GLYCOPROTEIN Ilia ON TEE PLATELET MEMBRANES

1987 ◽  
Author(s):  
S Niewiarowski ◽  
A Eckardt ◽  
H Lukasiewicz ◽  
K Norton ◽  
Tur-Fu Huanq ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Limit Of Detection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Apparent Molecular Weight ◽  
Progressive Increase ◽  
Partial Purification ◽  
Prolonged Storage ◽  
Granulocyte Elastase ◽  
Platelet Surface

Previous investigations from our laboratories have demonstrated the limited proteolysis by chymotrypsin of GPIIIa and the formation of a major component migrating on SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 60 kDa in a nonreduced system and 66 kDa in a reduced system. The formation of this component occurred in parallel with the express ion of fibrinogen receptors on the platelet surface. We raised in rabbits a monospecific polyclonal antibody against highly purified 60 kDa ccmponment. Using available rabbit polyclonal antisera (anti-GPIIIa and anti-66 kDa) and murine monoclonal anti GPIIIa antisera (AP3 and SSA6) a caiplete imnunological crossreactivity between GPIIIa and 60 kDa was established. The limit of detection by immunoblotting assay was 20 ng of GPIIIa or 60 kDa in Triton X100 extract of platelets. Extracts of intact platelets, of AEP- or thranbin-stEmulated platelets separated on SDS PAGE showed a strong band corresponding to GPIIIa, a light band migrating with an apparent molecular weight of 120 kDa and no detectable 60 kDa ccnponent. Incubation of platelets with chymotrypsin, pancreatic elastase or human granulocyte elastase resulted in the appearance of 60 kDa and in a progressive increase of 120 kDa component detectable by all polyclonal and monoclonal antisera. In contrast to 120 kDa, reduced GPIIIa (108 kDa) was not recognized by AP3 and SSA6. The 120 kDa did not dissociate in the presence of 6M urea and 5 mM EDTA. The component migrating with an apparent molecular weight of 120 kDa also appeared during prolonged storage of purified 60 kDa ccnponent. The partial purification of 60 kDa and 120 kDa components together with GPIIIa was accomplished by Con A sepharose chromatography. In conclusion 60 kDa and 120 kDa platelet membrane components represent products of proteolysis of GPIIIa.

scholarly journals Mouse macrophage elastase. Purification and characterization as a metalloproteinase

1981 ◽  
Vol 193 (2) ◽  
pp. 589-605 ◽  
Author(s):  
M J Banda ◽  
Z Werb
Keyword(s):  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
Serine Proteinases ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Predominant Form ◽  
Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

scholarly journals The release of collagenase as an inactive proenzyme by bone explants in culture

1972 ◽  
Vol 126 (2) ◽  
pp. 275-289 ◽  
Author(s):  
G. Vaes
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Inhibitor Complex ◽  
Ph Values ◽  
Liver Lysosomes ◽  
Skin Explants ◽  
Extracellular Activity

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme–inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a `procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.

scholarly journals Purification and Structural Characterization of a Novel Water-Soluble Neutral Polysaccharide from Cantharellus cibarius and Its Immunostimulating Activity in RAW264.7 Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Long Chen ◽  
Xichun Peng ◽  
Jiaying Lv ◽  
Siyin Liao ◽  
Shiyi Ou ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Functional Food ◽  
Peritoneal Macrophages ◽  
Water Soluble ◽  
Raw264.7 Cells ◽  
Molar Ratio ◽  
Immunostimulating Activity ◽  
Ft Ir ◽  
Mouse Peritoneal Macrophages

Polysaccharide is one of the important active ingredients of Cantharellus cibarius. The aims of this work were to analyze preliminary characterization and to investigate immunostimulating activity of a novel water-soluble neutral polysaccharide named JP1, which was purified from the fruiting body of Cantharellus cibarius using DEAE-FF chromatography and Sephadex G-100 chromatography. The characteristics of JP1 were determined by HPGPC, FT-IR spectra, gas chromatography, and Congo Red Method. Immunostimulating activity of JP1 was investigated in RAW264.7 cells. Results indicated that JP1 consisted of L-Arabinose, D-Mannose, D-Glucose, and D-Galactose in a molar ratio of 1 : 1.06 : 1.95 : 1.17 with a molecular weight of 336 kDa. JP1 is nontoxic to RAW264.7 cells at this concentration range (62.5–1000 μg/mL). Furthermore, JP1 can promote mouse peritoneal macrophages to secrete NO and enhance the secretion of macrophages’ cytokines IL-6 in RAW264.7 cells. These results suggested that JP1 could have potential immunostimulating activity applications as medicine or functional food.

scholarly journals Identification of cytoskeletal components involved in attachment of L929 cells and macrophages to polystyrene.

1981 ◽  
Vol 89 (3) ◽  
pp. 691-694 ◽  
Author(s):  
K W Lanks ◽  
N W Chin
Keyword(s):  
Molecular Weight ◽  
Peptide Mapping ◽  
Limited Proteolysis ◽  
Detailed Examination ◽  
Peritoneal Macrophages ◽  
Intimate Contact ◽  
L929 Cells ◽  
Molecular Weights ◽  
Murine Peritoneal Macrophages ◽  
Triton X

We have previously shown that lactoperoxidase (LPO) covalently coupled to polystyrene tissue culture flasks can be used to radioiodinate monolayer cell proteins that come into intimate contact with the LPO-polystyrene surface. These studies have now been extended to include a detailed examination of the class of iodinated polypeptides migrating with apparent molecular weights of 50,000 and 55,000 in SDS polyacrylamide gels. Whereas in cultured L929 cells the 55,000 band is predominantly iodinated, in thioglycollate-activated murine peritoneal macrophages the 55,000 and 50,000 bands are of equal intensity. It is possible that the marked degree of exposure of the 50,000 mol wt polypeptide to immobilized LPO is related to the unique strength of macrophages attachment. After labeling of both L929 cells and macrophages with immobilized LPO, all polypeptides in this molecular weight region were subjected to peptide mapping by simultaneous limited proteolysis and electrophoresis in a second SDS polyacrylamide slab gel. The results clearly show that the two major polypeptides in this region are identical within the limits of resolution of this technique. The 55,000 mol wt polypeptide can also be identified in Triton X-100 cytoskeletons from L929 cells after labeling with soluble LPO either before or after detergent lysis. We conclude that this cell surface polypeptide is in continuity with the cytoskeleton and is preferentially exposed to the substratum during attachment to polystyrene.

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