scholarly journals The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

1978 ◽  
Vol 172 (2) ◽  
pp. 261-274 ◽  
Author(s):  
G Vaes ◽  
Y Eeckhout ◽  
G Lenaers-Claeys ◽  
C François-Gillet ◽  
J E Druetz
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Neutral Proteinase ◽  
Inhibitor Complex ◽  
The Media ◽  
Parallel Activation ◽  
Cartilage Proteoglycans

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

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 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oils from AgNPs and AuNPs Elicited Lavandula angustifolia In Vitro Cultures

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 606 ◽  
Author(s):  
Aneta Wesołowska ◽  
Paula Jadczak ◽  
Danuta Kulpa ◽  
Włodzimierz Przewodowski
Keyword(s):  
Molecular Weight ◽  
Silver Nanoparticles ◽  
Essential Oils ◽  
Culture Media ◽  
In Vitro Cultures ◽  
Gas Chromatography Mass Spectrometry ◽  
Lavandula Angustifolia ◽  
Gold And Silver Nanoparticles ◽  
The Media

The aim of this study was to determine how the addition of gold and silver nanoparticles to culture media affects the composition of essential oils extracted from Lavandula angustifolia propagated on MS media with the addition of 10 and 50 mg·dm−3 of gold (24.2 ± 2.4 nm) and silver (27.5 ± 4.8 nm) nanocolloids. The oil extracted from the lavender tissues propagated on the medium with 10 mg·dm−3 AgNPs (silver nanoparticles) differed the most with respect to the control; oil-10 compounds were not found at all, and 13 others were detected which were not present in the control oil. The addition of AuNPs (gold nanoparticles) and AgNPs to the media resulted in a decrease of lower molecular weight compounds (e.g., α- and β-pinene, camphene, δ-3-carene, p-cymene, 1,8-cineole, trans-pinocarveol, camphoriborneol), which were replaced by those of a higher molecular weight (τ- and α-cadinol 9-cedranone, cadalene, α-bisabolol, cis-14-nor-muurol-5-en-4-one, (E,E)-farnesol).

scholarly journals Screening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds

2010 ◽  
Vol 15 ◽  
pp. 19
Author(s):  
Maher Ali. Maqtari ◽  
A.B. Mohamed. Saad
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Apparent Dissociation Constant ◽  
Chymotrypsin Inhibitor ◽  
Ammonium Sulphate Precipitation ◽  
Wide Ph Range ◽  
Ph Range

A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman  (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme-  inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2  groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C. 

scholarly journals The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor

1983 ◽  
Vol 211 (2) ◽  
pp. 313-318 ◽  
Author(s):  
T E Cawston ◽  
G Murphy ◽  
E Mercer ◽  
W A Galloway ◽  
B L Hazleman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Concanavalin A ◽  
Culture Medium ◽  
Enzyme Inhibitor ◽  
Inhibitor Complex ◽  
Metalloproteinase Inhibitor ◽  
Rabbit Bone

1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 × 10(-10) M. 2. The molecular weight of the enzyme-inhibitor complex was found to be 54 000, but no enzyme activity could be recovered from the complex after treatment with either mercurials or proteinases. The complex thus differed from latent collagenase in terms of size, susceptibility to mercurials and behaviour on concanavalin A-Sepharose. 3. The interaction of the purified components was compared with that of crude collagenase and crude inhibitor in culture medium. Mercurial treatment partially reversed the inhibition in the crude system, but not when the purified components were used. 4. The significance of the results is discussed in relation to the extracellular control of the activity of collagenase.

Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

scholarly journals A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein

1972 ◽  
Vol 129 (1) ◽  
pp. 141-152 ◽  
Author(s):  
I. H. Mather ◽  
M. Knight
Keyword(s):  
Pseudomonas Putida ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Inhibitory Protein ◽  
Inhibitor Complex ◽  
Molecular Weights ◽  
Heat Stable ◽  
Culture Supernatants

A thermostable NAD(P)+glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.

scholarly journals Degradation of cartilage proteoglycans by a neutral proteinase secreted by rabbit bone-marrow macrophages in culture

1978 ◽  
Vol 172 (2) ◽  
pp. 275-284 ◽  
Author(s):  
P Hauser ◽  
G Vaes
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Culture Media ◽  
Neutral Proteinase ◽  
Inflammatory Processes ◽  
Joint Erosions ◽  
Rabbit Bone ◽  
Cartilage Proteoglycans

When cultivated together with pieces of cartilage biosynthetically labelled with 35S in their proteoglycans, rabbit macrophages, differentiated in vitro from bone-marrow cells, cause the release of soluble 35S-labelled material into the culture medium. This process is inhibited by killing the macrophages or by cycloheximide treatment, and is due to the secretion by the cells of a metal-dependent neutral proteinase capable of degrading cartilage proteoglycan subunits into fragments of high molecular weight. Enzyme activity is optimum at about pH7, and is inhibited by EDTA, o-phenanthroline, cysteine or serum, but not by di-isopropyl phosphorofluoridate nor by 4-hydroxymercuribenzoate. The effect of EDTA is partially reversed by Co2+ or Zn2+ ions. The enzyme is eluted from Sephadex G-150 columns as a single peak of material (apparent mol.wt. 17000) that contains also most of the proteolytic activity exerted by culture media on Azocoll (denatured collagen) or on casein. The possible role of this metalloproteinase in chronic inflammatory processes is discussed, particularly in connection with joint erosions in rheumatoid arthritis.

Renin in the Mouse Kidney Has a Molecular Weight of 40 000

1981 ◽  
Vol 60 (1) ◽  
pp. 41-46 ◽  
Author(s):  
K. Poulsen ◽  
A. H. Nielsen
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
High Molecular Weight ◽  
Enzyme Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Antigenic Properties ◽  
Before And After

1. Mouse kidney was homogenized in a mixture of serine-metallo- and thiol-enzyme inhibitors. The homogenate proteins were separated with respect to size and charge by gel filtration, agarose electrophoresis and polyacrylamide-gel electrophoresis. 2. Renin was localized by its enzymatic activity by using the antibody trapping radioimmunoassay for angiotensin I, before and after acid treatment and limited proteolysis. Renin was also localized by its antigenic properties by using antirenin antibodies elicited against pure mouse submaxillary renin. The antibody cross-reacted fully with mouse kidney renin and with high-molecular-weight renin forms in mouse plasma. 3. In the kidney only fully enzymically active 40 000 renin could be detected enzymically and antigenically. No high-molecular-weight renin or inactive renin was demonstrable. 4. Two electrophoretically different renin forms were seen in accordance with renin being a glycoprotein. They were both fully enzymically active with identical specific enzymatic activities. 5. The mouse kidney renin had a specific enzymatic activity identical with that of pure mouse submaxillary renin, being 0.4 Goldblatt unit/μg.

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