Purification and structural characterization of a cartilage matrix protein

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.

Download Full-text

A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

Biochemical Journal ◽

10.1042/bj1970355 ◽

1981 ◽

Vol 197 (2) ◽

pp. 355-366 ◽

Cited By ~ 97

Author(s):

D Heinegård ◽

M Paulsson ◽

S Inerot ◽

C Carlström

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Chondroitin Sulphate ◽

Polyacrylamide Gel Electrophoresis ◽

Buoyant Density ◽

Sodium Dodecyl ◽

Partial Specific Volume ◽

Gel Chromatography ◽

Guanidinium Chloride ◽

Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

Download Full-text

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains

Biochemical Journal ◽

10.1042/bj1830325 ◽

1979 ◽

Vol 183 (2) ◽

pp. 325-330 ◽

Cited By ~ 18

Author(s):

E Ilan ◽

E Daniel

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Sedimentation Equilibrium ◽

Guanidinium Chloride ◽

Tadpole Shrimp ◽

Polypeptide Chains

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.

Download Full-text

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage

Biochemical Journal ◽

10.1042/bj1830539 ◽

1979 ◽

Vol 183 (3) ◽

pp. 539-545 ◽

Cited By ~ 91

Author(s):

M Paulsson ◽

D Heinegård

Keyword(s):

High Speed ◽

Matrix Protein ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sucrose Density Gradient ◽

Exchange Chromatography ◽

Cartilage Matrix ◽

Gel Chromatography ◽

Sucrose Density Gradient Centrifugation

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.

Download Full-text

Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans

Biochemical Journal ◽

10.1042/bj2010027 ◽

1982 ◽

Vol 201 (1) ◽

pp. 27-37 ◽

Cited By ~ 69

Author(s):

C H Pearson ◽

G J Gibson

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Periodontal Ligament ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Sedimentation Equilibrium ◽

Gel Chromatography ◽

Dermatan Sulphate ◽

Self Association

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.

Download Full-text

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

Canadian Journal of Biochemistry ◽

10.1139/o77-143 ◽

1977 ◽

Vol 55 (9) ◽

pp. 958-964 ◽

Cited By ~ 6

Author(s):

M. P. C. Ip ◽

R. J. Thibert ◽

D. E. Schmidt Jr.

Keyword(s):

Molecular Weight ◽

Rat Liver ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Liver Homogenate ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Labile Hydrogen ◽

Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Download Full-text

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits

Biochemical Journal ◽

10.1042/bj2070297 ◽

1982 ◽

Vol 207 (2) ◽

pp. 297-303 ◽

Cited By ~ 24

Author(s):

E Ilan ◽

E Weisselberg ◽

E Daniel

Keyword(s):

Molecular Weight ◽

Daphnia Magna ◽

Quaternary Structure ◽

Slow Component ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Sedimentation Equilibrium ◽

Subunit Structure ◽

Single Chain

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.

Download Full-text

A study of equilibrium binding of link protein to hyaluronate

Biochemical Journal ◽

10.1042/bj2130445 ◽

1983 ◽

Vol 213 (2) ◽

pp. 445-450 ◽

Cited By ~ 12

Author(s):

M Lyon ◽

I A Nieduszynski

Keyword(s):

Dissociation Constants ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Molecule ◽

Gel Chromatography ◽

Guanidinium Chloride ◽

Link Protein ◽

Equilibrium Binding ◽

Femoral Head Cartilage

Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.

Download Full-text

The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

Biochemical Journal ◽

10.1042/bj1970427 ◽

1981 ◽

Vol 197 (2) ◽

pp. 427-436 ◽

Cited By ~ 9

Author(s):

G A Nimmo ◽

J R Coggins

Keyword(s):

Molecular Weight ◽

Neurospora Crassa ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Polyacrylamide Gel ◽

Sedimentation Equilibrium ◽

Phosphate Synthase

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.

Download Full-text

The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

Biochemical Journal ◽

10.1042/bj1370489 ◽

1974 ◽

Vol 137 (3) ◽

pp. 489-495 ◽

Cited By ~ 125

Author(s):

M. A. Kerr ◽

A. J. Kenny

Keyword(s):

Molecular Weight ◽

Brush Border ◽

Sodium Dodecyl Sulphate ◽

Guanidine Hydrochloride ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Atomic Absorption Spectrophotometry ◽

Sedimentation Equilibrium ◽

Rabbit Kidney

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [125I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn2+ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn2+- activated endopeptidase to be characterized.

Download Full-text

Isolation and characterization of senile amyloid--related antigenic substance (SASSAM) from mouse serum. Apo SASSAM is a low molecular weight apoprotein of high density lipoprotein.

Journal of Experimental Medicine ◽

10.1084/jem.158.5.1600 ◽

1983 ◽

Vol 158 (5) ◽

pp. 1600-1614 ◽

Cited By ~ 26

Author(s):

K Higuchi ◽

A Matsumura ◽

K Hashimoto ◽

A Honma ◽

S Takeshita ◽

...

Keyword(s):

Molecular Weight ◽

High Density Lipoprotein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Density Lipoprotein ◽

High Density ◽

Mouse Serum ◽

Gel Chromatography ◽

Precipitation Line ◽

Isolation And Characterization

Sera obtained from senescence-accelerated mouse (SAM) and normal mice contained a substance that reacted with antiserum raised against ASSAM, a novel senile amyloid fibril protein isolated from the liver of SAM. This physiological substance, termed "SASSAM" (serum ASSAM-related antigenic substance), migrated to the albumin/prealbumin region in immunoelectrophoresis and the precipitation line formed with anti-ASSAM antiserum was stained positively with both Amide Black 10 B and Oil Red O/Fat Red 7B solutions, thereby suggesting that SASSAM is an alpha lipoprotein. Using Sephadex G-200 gel chromatography, SASSAM was eluted as a high mol wt form of approximately 200,000 daltons. Fractionation of lipoprotein from normal mouse serum by preparative ultra-centrifugation disclosed that SASSAM was found mainly in high density lipoprotein, HDL (the density is between 1.063 and 1.21 g/cm3). The largest amount of SASSAM was found in the HDL2 fraction (the density is between 1.063 and 1.125) and in this fraction SAA was not detected. Furthermore, ASSAM immunoreactivity appeared in the low mol wt proteins (below 10,000 daltons) of apo HDL separated in the buffer containing 8 M urea through Sephadex G-200. In 8 M urea sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the major components of apolipoproteins in this position, possibly corresponding to apo C proteins, have the same molecular weight, 5,200 daltons, as ASSAM and this component was labeled by anti-ASSAM antiserum after transfer to nitrocellulose paper.

Download Full-text