Isolation And Purification Of Collagen And α1 Receptor From Platelet Membrane

1981 ◽  
Author(s):  
T M Chiang ◽  
A H Kang
Keyword(s):  
Affinity Chromatography ◽  
Binding Site ◽  
Gel Electrophoresis ◽  
Type I Collagen ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Type I ◽  
Isolation And Purification ◽  
Platelet Membranes

We have previously demonstrated that chick skin type I collagen and the α1(I) chain mediate platelet aggregation. Aggregation is associated with specific binding of these substances by platelet membranes. We now describe the isolation and purification of the receptor. Platelet membranes were prepared as described previously and isolated membranes were solubilized in 0.5% Triton. The receptor was then purified by a combination of gel filtration, affinity chromatography on α1(I)-sepharose or type I collagen-sepharose and preparative polyacrylamide gel electrophoresis. The receptor activity was assayed either directly by a binding assay using (14C)-glycine-labeled α1(I) or indirectly by an adhesion inhibition assay on Sepharose 2B with (14C)-sero- tinin-labeled platelets.The results show that the α1(I) receptor can be purified to a single band on SDS-gel electrophoresis with a recovery of 2.5%. Its activity is destroyed by preincubation with trypsin or pronase indicating it is a protein. The apparent molecular weight as estimated by gel filtration and SDS-gel electrophoresis is 95,000 daltons. The binding of (14C)- labeled α1(I) is specifically displaced by unlabeled α1(I), and the bound radioactivity can be removed by treatment with purified bacterial collagenase. The binding of (14C)- labeled α1(I) by the purified α1(I) receptor can also be inhibited by the receptor isolated from collagen-sepharose affinity chromatography. These data suggest that the α1(I) binding site is identical to the collagen binding site.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

A three step purification procedure for human liver ferritin

1980 ◽  
Vol 58 (6) ◽  
pp. 494-498 ◽  
Author(s):  
M. Pagé ◽  
J. Lagueux ◽  
C. Gauthier
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Human Liver Ferritin ◽  
Normal Human Liver ◽  
Normal Human ◽  
Liver Ferritin

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.

scholarly journals Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen

1976 ◽  
Vol 153 (2) ◽  
pp. 259-264 ◽  
Author(s):  
V Lee-Own ◽  
J C Anderson
Keyword(s):  
Gel Electrophoresis ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Type Ii Collagen ◽  
Type I ◽  
Type Ii ◽  
Nasal Cartilage ◽  
Preferential Binding ◽  
Calf Skin

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.

scholarly journals Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content

1980 ◽  
Vol 189 (1) ◽  
pp. 111-124 ◽  
Author(s):  
N D Light ◽  
A J Bailey
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Type I ◽  
Molecular Weight Range ◽  
Carbohydrate Analysis ◽  
Weight Range

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.

Purification of Porcine and Human Tissue Activator

1979 ◽  
Author(s):  
P Wallen ◽  
M Rånby ◽  
N Bergsdorf ◽  
P Kok
Keyword(s):  
Affinity Chromatography ◽  
Human Tissue ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Potassium Thiocyanate ◽  
Polyacrylamide Gel ◽  
Main Band ◽  
Reference Preparation

Tissue activator from pig heart: A highly purified activator preparation from pig heart has been prepared, essentially using two affinity adsorbtion steps. 1) Affinity adsorbtion to fibrin and elution with potassium thiocyanate. 2) Affinity chromatography on Sepharose-arginine. The final product, which is obtained by gel filtration on Sephacryl S-200 contains according to SDS-polyacrylamide gel electrophoresis one main band with Mw 64000. Reduced samples still appear as one component but with Mw 31000. The specific activity is about 500000 IU/mg (WHO Reference Preparation for Urokinase) and the yield 15-25 %. Tissue activator from human uterus: A highly purified preparation of human tissue activator has been prepared from uterus by an immunosorbent technique using antibodies produced in goats against the porcine tissue activator and coupled to Sepharose. A crude preparation from 1 kg uterus tissue and containing about 100000 IU tissue activator was adsorbed on 30 g of the immunosorbent. The activity was eluted with a KSCN-gradient. Further purification was obtained by affinity chromatography on Sepharose-arginine. The yield in the active fraction was 30-35 % and the specific activity 200000 to 300000 IU/mg. SDS-polyacrylamide gel electrophoresis showed one main band and 1-2 additional trace components.

The purification of porcine haptoglobin by affinity chromatography with concanavalin A – Sepharose

1976 ◽  
Vol 54 (11) ◽  
pp. 999-1001 ◽  
Author(s):  
Frank A. Terpstra ◽  
David B. Smith
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Small Scale ◽  
Scale Separation

Methods designed for the isolation of human haptoglobin (Hp) were found insufficient when applied to pig plasma due to the formation of a material tentatively identified as albumin dimer. Small scale separation is possible by preparative polyacrylamide gel electrophoresis. Larger scale purification from nonglycoprotein contaminants such as albumin dimer is achieved by affinity chromatography using immobilized concanavalin A. Porcine haptoglobin is microheterogeneous. More than 14 components, partially resolveable by gel filtration, were detected.

Inhibition of Urokinase by Complex Formation with Human Antithrombin III in Absence and Presence of Heparin

1978 ◽  
Vol 39 (03) ◽  
pp. 616-563 ◽  
Author(s):  
Inge Clemmensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Crossed Immunoelectrophoresis

SummaryHuman antithrombin III was purified from fresh human plasma by affinity chromatography on heparin-Sepharose®, affinity chromatography on concanavalin A Sepharose®, gel filtration on Ultrogel® AcA 34, ion exchange chromatography on DEAE A-50 Sephadex® and preparative agarose gel electrophoresis. The hydrolytic activity of urokinase (plasminogen activator from urine) on acetyl-glycyl-L-lysine methyl ester acetate (Ac-gly-lys-OMe Ac) was inhibited by antithrombin III in a slow time-dependent manner. Heparin accelerated the reaction between activator and inhibitor. Inhibition of catalytic activity was associated with the formation of an 1:1 molar complex between activator and inhibitor as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The complex was also demonstrated by crossed Immunoelectrophoresis against anti-antithrombin III.

scholarly journals Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

1977 ◽  
Vol 166 (3) ◽  
pp. 437-446 ◽  
Author(s):  
J. David Schofield ◽  
Richard Harwood ◽  
David S. Jackson
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Helical Conformation ◽  
Type I ◽  
Embryonic Chick ◽  
Gel Filtration Chromatography ◽  
Free Cells ◽  
Matrix Free

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [14C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[14C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-α-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.

scholarly journals Autophagy facilitates type I collagen synthesis in periodontal ligament cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Nakamura ◽  
Motozo Yamashita ◽  
Kuniko Ikegami ◽  
Mio Suzuki ◽  
Manabu Yanagita ◽  
...  
Keyword(s):  
Periodontal Ligament ◽  
Collagen Synthesis ◽  
Type I Collagen ◽  
Periodontal Diseases ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Physiological Role ◽  
Type I ◽  
Periodontal Tissue ◽  
Pdl Cells

AbstractAutophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

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