scholarly journals Cathepsin B1. A lysosomal enzyme that degrades native collagen

1974 ◽  
Vol 137 (2) ◽  
pp. 387-398 ◽  
Author(s):  
Mary C. Burleigh ◽  
Alan J. Barrett ◽  
Gerald S. Lazarus
Keyword(s):  
Optical Rotation ◽  
Gel Filtration ◽  
Collagen Fibrils ◽  
Ph Optimum ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Acid Soluble Collagen ◽  
Lysosomal Proteinases ◽  
Native Collagen ◽  
Helical Region

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released 14C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human α2-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5–5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an α-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.

scholarly journals The effect of ultraviolet irradiation on soluble collagen

1965 ◽  
Vol 97 (1) ◽  
pp. 139-147 ◽  
Author(s):  
DR Cooper ◽  
RJ Davidson
Keyword(s):  
Ultraviolet Irradiation ◽  
Optical Rotation ◽  
Gel Filtration ◽  
Collagen Molecule ◽  
Neutral Salt ◽  
Soluble Collagen ◽  
Acid Soluble Collagen ◽  
Skin Collagen ◽  
Kinetics Of ◽  
Calf Skin

1. The effect of ultraviolet irradiation on acid-soluble and neutral-salt-soluble calf-skin collagen was studied by chromatography, gel filtration, amino acid analysis and sedimentation of the sub-units, and the reaction kinetics of degradation were obtained from viscosity and optical rotation measurements. 2. It was demonstrated that, whereas the structure of neutral-salt-soluble calf-skin collagen may be represented by the formula (alpha(1))(2)alpha(2), the acid-soluble extract has the formula alpha(1).(alpha(2))(2). The acid-soluble collagen is also unusual in containing a large amount of a component that could be beta(22). 3. Ultraviolet irradiation causes the progressive degradation of the collagen molecule into smaller molecular fragments that subsequently lose their helical nature. The rate constants show that the denaturation of soluble collagens by ultraviolet irradiation is much slower, under the conditions used, than denaturation by heat or enzymes.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals The effect of semicarbazide on the nature and stability of collagen fibrils

1970 ◽  
Vol 118 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Shirley Ayad ◽  
C. H. Wynn
Keyword(s):  
Collagen Fibril ◽  
Collagen Fibrils ◽  
Cross Linking ◽  
Unsaturated Aldehyde ◽  
Cross Link ◽  
Soluble Collagen ◽  
Acid Soluble Collagen ◽  
Direct Binding ◽  
Aldehyde Groups

Semicarbazide affected both the final width and stability of fibrils reconstituted from solutions of acid-soluble collagen. Fibril width was increased after semicarbazide treatment at pH2.6 and 4.3, whereas after treatment at pH8.9 it decreased. Fibril stability was decreased after semicarbazide treatment at all values of pH and temperature, indicating the inhibition of intermolecular cross-linking. A direct binding of semicarbazide to the αβ-unsaturated aldehyde groups in the intramolecular cross-link was demonstrated.

scholarly journals Partial purification and characterization of collagen galactosyltransferase from chick embryos

1976 ◽  
Vol 155 (1) ◽  
pp. 145-153 ◽  
Author(s):  
L Risteli ◽  
R Myllyä ◽  
K I Kivirikko
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Helical Conformation ◽  
Tissue Homogenate ◽  
Partial Purification ◽  
Molecular Weights ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Chick Embryo Extract ◽  
Triton X

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.

scholarly journals The decomposition of soluble collagen by γ-irradiation

1969 ◽  
Vol 113 (2) ◽  
pp. 263-269 ◽  
Author(s):  
D. R. Cooper ◽  
A. E. Russell
Keyword(s):  
Irradiation Dose ◽  
Optical Rotation ◽  
Specific Rotation ◽  
Γ Irradiation ◽  
Soluble Collagen ◽  
Heating And Cooling ◽  
Reduced Viscosity ◽  
Acid Soluble Collagen ◽  
Freeze Dried ◽  
High Degree

1. The effect of γ-irradiation in the range 1 krad–10 Mrads on freeze-dried acid-soluble collagen was studied. 2. The specific-rotation and reduced-viscosity recoveries after heating and cooling of the irradiated collagen in solution showed a high degree of dependence on irradiation dose, with reduced viscosity showing significantly less recovery than specific rotation on increasing the irradiation dose. 3. The dependence of reduced viscosity on concentration was greatly decreased with increased doses of γ-irradiation. 4. The melting temperature measured by optical rotation also decreased as the irradiation dose was increased, and at low doses was distinctly biphasic. 5. Physical properties showed that the action of γ-irradiation up to 10 Mrads occurred in two distinct phases, with the early changes being extremely sensitive to irradiation dose. 6. The action of the γ-irradiation is discussed in terms of the structure of tropocollagen.

scholarly journals Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1970 ◽  
Vol 118 (2) ◽  
pp. 229-232 ◽  
Author(s):  
Chisato Hirayama ◽  
Ikuo Morotomi ◽  
Kaichiro Hiroshige
Keyword(s):  
Carbon Tetrachloride ◽  
Rat Liver ◽  
Collagenolytic Activity ◽  
Normal Range ◽  
Carbon Tetrachloride Poisoning ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Acid Soluble Collagen ◽  
Soluble Acid

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [14C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./μmol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.

scholarly journals Effect of pH on thermal stability of collagen in the dispersed and aggregated states (Short Communication)

1974 ◽  
Vol 139 (1) ◽  
pp. 277-280 ◽  
Author(s):  
Allan E. Russell
Keyword(s):  
Thermal Stability ◽  
Short Communication ◽  
Dilute Solution ◽  
Thermal Stabilities ◽  
Effect Of Ph ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Acid Soluble Collagen ◽  
Collagen Molecules ◽  
Thermal Stability Of

Thermal stabilities of mature insoluble collagen, salt-precipitated fibrils of acid-soluble collagen and acid-soluble collagen in solution were compared as a function of acid pH. Both insoluble and precipitated collagens showed large parallel destabilization with decrease in pH, whereas the intrinsic stability of individual collagen molecules in dilute solution was comparatively unaffected.

Banded Structures in the Connective Tissue of Meningioma

1976 ◽  
Vol 34 ◽  
pp. 234-235
Author(s):  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Connective Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Lead Citrate ◽  
Connective Tissues ◽  
Native Collagen ◽  
Routine Procedures

Previously, we have reported on extracellular cross-striated banded structures in human connective tissues of a variety of organs (1). Since then, more material has been examined and other techniques applied. Recently, we studied a fibrocytic meningioma of the falx. After the specimen was fixed in 4% buffered glutaraldehyde and post-fixed in 1% buffered osmium tetroxide, other routine procedures were followed for embedding in Epon 812. Sections were stained with uranyl acetate and lead citrate. There were numerous cross striated banded structures in aggregated bundle forms found in the connecfive tissue of the tumor. The banded material has a periodicity of about 450 Å and where it assumes a filamentous arrangement, appears to be about 800 Å in diameter. In comparison with the vicinal native collagen fibrils, the banded material Is sometimes about twice the diameter of native collagen.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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