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 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 Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase

1990 ◽  
Vol 269 (1) ◽  
pp. 183-187 ◽  
Author(s):  
T E Cawston ◽  
V A Curry ◽  
I M Clark ◽  
B L Hazleman
Keyword(s):  
Connective Tissue ◽  
Culture Media ◽  
Tight Binding ◽  
Gel Filtration ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Metalloproteinase Inhibitor ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Extracellular Activity

Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named ‘large inhibitor of metalloproteinases’ (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.

The Effect of Leucocyte Elastase on the Immunoelectrophoretic Behaviour of α1-Antitrypsin

1984 ◽  
Vol 66 (2) ◽  
pp. 217-224 ◽  
Author(s):  
R. A. Stockley ◽  
S. C. Afford
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Enzyme Inhibitor ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Two Dimensional ◽  
Ph Values ◽  
Inhibitory Capacity ◽  
Leucocyte Elastase ◽  
Enzyme Complexes

1. Two-dimensional immunoelectrophoresis and conventional sodium dodecyl sulphate-polyacryl-amide gel electrophoresis was performed on various mixtures of purified α1-antitrypsin (α1AT) and leucocyte elastase (LE). 2. The results confirm that α1AT inhibits LE by the formation of enzyme-inhibitor complexes demonstrable by both techniques. 3. The complexes break down with time and are not affected by pH in the presence of excess α1AT. However, the breakdown is more rapid in the presence of excess enzyme only at pH values where LE remains active. The resultant products of the complex breakdown include inactivated LE and α1AT that has undergone limited proteolysis. 4. It is concluded that the presence or absence of α1AT-enzyme complexes as demonstrated by two-dimensional immunoelectrophoresis must be interpreted with caution when studying α1AT function in lung secretions. The absence of such complexes does not mean that previous interaction with enzyme has not occurred, thereby accounting for a reduction in α1AT inhibitory capacity.

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. 

Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021

2019 ◽  
Author(s):  
Elham Noroozi ◽  
Naheed Mojgani ◽  
Elahe Motevaseli ◽  
Mohammad Hossein Modarressi ◽  
Majid Tebianian
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Casei ◽  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Shigella Flexneri ◽  
Gel Filtration ◽  
Antimicrobial Protein ◽  
Purification Method ◽  
Ph Values ◽  
Subsequent Decrease

Background and Objectives: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by au- tochthonous Lactobacillus casei TA0021 strain. Materials and Methods: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. Results: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of ˃9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. Conclusion: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

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.

Sign in / Sign up

Export Citation Format

Share Document