scholarly journals Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin

1981 ◽  
Vol 199 (3) ◽  
pp. 807-811 ◽  
Author(s):  
G Murphy ◽  
T E Cawston ◽  
W A Galloway ◽  
M J Barnes ◽  
R A D Bunning ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Culture Medium ◽  
Type Iv Collagen ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Bone Culture ◽  
Gel Filtration Chromatography ◽  
Type Iv ◽  
Rabbit Bone ◽  
Type V

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.

scholarly journals Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components

1983 ◽  
Vol 209 (3) ◽  
pp. 741-752 ◽  
Author(s):  
W A Galloway ◽  
G Murphy ◽  
J D Sandy ◽  
J Gavrilovic ◽  
T E Cawston ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Culture Medium ◽  
Type Iv Collagen ◽  
Sodium Dodecyl ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Type Iii Collagen ◽  
Bone Culture ◽  
Nasal Cartilage ◽  
Type Iv ◽  
Rabbit Bone

A metalloproteinase, ‘proteoglycanase’, that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the ‘weak region’ of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.

DNA Polymerase Activity Associated with Endogenous Template: Release by Ribonuclease Treatment

1975 ◽  
Vol 53 (5) ◽  
pp. 574-582
Author(s):  
Francesco Moranelli ◽  
P. E. Penner
Keyword(s):  
Molecular Weight ◽  
Dna Polymerase ◽  
Gel Filtration ◽  
Polymerase Activity ◽  
Void Volume ◽  
Lower Molecular Weight ◽  
Single Peak ◽  
Gel Filtration Chromatography ◽  
Thymus Extract ◽  
Concomitant Decrease

A ribonuclease-sensitive DNA polymerase, which uses an endogenous template, is detectable in the 39 000 g supernatant of a rat thymus homogenate, and appears as a single peak of activity in the void volume after Sephadex G 150 or G 200 gel filtration chromatography. Native and "activated" DNA-dependent DNA polymerase activities coincide with the endogenous-templated polymerase activity. Treatment of the thymus extract with ribonuclease(s) prior to gel filtration chromatography yields two other peaks of activity in addition to the void volume peak. The appearance of the two lower molecular weight peaks of activity is accompanied by a concomitant decrease in the endogenous-templated activity. The effect of ribonuclease is specific and cannot be reproduced by a similar deoxyribonuclease treatment.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

scholarly journals Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

1984 ◽  
Vol 98 (5) ◽  
pp. 1637-1644 ◽  
Author(s):  
R Mayne ◽  
H Wiedemann ◽  
M H Irwin ◽  
R D Sanderson ◽  
J M Fitch ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cleavage Site ◽  
Type Iv Collagen ◽  
Collagen Molecule ◽  
Electron Microscopic ◽  
Type V Collagen ◽  
Type Iv ◽  
Type V ◽  
Rotary Shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.

scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

scholarly journals Difference in Thrombolytic Effect between Higher and Lower Molecular Weight Forms of Urokinase

1977 ◽  
Author(s):  
T. Suyama ◽  
M. Nishida ◽  
Y. Iga ◽  
R. Naito
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Dissolution Time ◽  
Thrombolytic Activity ◽  
Lysis Time ◽  
Thrombolytic Effect ◽  
Approximate Molecular Weight ◽  
Made In

Urokinase (UK) from human urine has been widely used for thrombolytic therapy in Japan. However, commercially available preparations are not identical but consist of mainly two forms of UK with higher and lower molecular weight (H-UK and L-UK) . An attempt was made in this report to compare thrombolytic activity of H-UK with that of L-UK on artificial thrombi produced from human blood by a modification of Chandler’s loop method, which was somehow comparable to the situation in vivo. Two active forms of UK were purified from crude preparation by gel filtration. The approximate molecular weight of the H-UK was 54,000 and of the L-UK 34,000. The potency of UK was determined by “two-stage lysis time method” and expressed by International unit(lU). Thrombolytic activity measured by Chandler’s method was calculated as % lysis of the control thrombus that was formed in the abscence of UK.As a result, thrombus-dissolution time of H-UK was much shorter than that of L-UK. Furthermore, concentration of H-UK (IU/ml blood) necessary to induce 50% lysis was approximately one half lower than that of L-UK. The similiar results were obtained on artificial thrombi from the blood of dog, rat and rabbit. The data suggest that H-UK seems to be more effective on treatment of thromboembolicdisorders as compared to L-UK in terms of the same IU basis.

A Potent Insect Chitinase Inhibitor of Fungal Origin

2003 ◽  
Vol 58 (11-12) ◽  
pp. 891-894 ◽  
Author(s):  
Teruhiko Nitoda ◽  
Hirokazu Usuki ◽  
Hiroshi Kanzaki
Keyword(s):  
Molecular Weight ◽  
Anion Exchange ◽  
Culture Filtrate ◽  
Inhibitory Activity ◽  
Spodoptera Litura ◽  
Gel Filtration ◽  
Fungal Strain ◽  
Water Soluble ◽  
Gel Filtration Chromatography ◽  
Soluble Polysaccharide

Abstract A water-soluble polysaccharide was isolated from the culture filtrate of a fungal strain, Sphaeropsis sp. TNPT116-Cz, as a novel insect chitinase inhibitor. It was purified to chromatographic homogeneity by ethanol precipitation, anion-exchange and gel filtration chromatography. Its molecular weight was estimated to be 16 kDa by gel filtration HPLC. Monosaccharide analysis showed that it contained glucose, galactose, N-acetylglucosamine and a deoxysugar. This polysaccharide showed potent and specific inhibitory activity against Spodoptera litura chitinase with an IC50 value of 28 nᴍ.

Macromolecular constituents of basement membranes: a review of current knowledge on their structure and function

1983 ◽  
Vol 61 (8) ◽  
pp. 942-948 ◽  
Author(s):  
Paul G. Scott
Keyword(s):  
Type Iv Collagen ◽  
Current Knowledge ◽  
Heparan Sulphate ◽  
Structural Features ◽  
Structure And Function ◽  
Basement Membranes ◽  
Type Iv ◽  
Type V ◽  
And Function ◽  
Additional Form

Macromolecules which appear to be integral constituents of basement membranes include type IV collagen, the glycoprotein laminin, and heparan sulphate proteoglycan. Another glycoprotein, fibronectin, may occupy an intermediate position between some lining cells and their basement membranes but is not, however, restricted to this location. An additional form of collagen, genetic type V which differs significantly from type IV collagen in structure, appears to be associated with some basement membranes, possibly linking them to underlying connective tissue. The main structural features of each of these macromolecules, as presently understood, are reviewed here as a background to the experimental papers in this "mini-symposium."

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