Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Hydrodynamic and optical properties of the wheat germ Em protein

1985 ◽  
Vol 63 (8) ◽  
pp. 803-811 ◽  
Author(s):  
William D. McCubbin ◽  
Cyril M. Kay ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Gel Filtration ◽  
Fluorescence Emission ◽  
Sedimentation Coefficient ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Partial Specific Volume ◽  
Fluorescence Emission Spectrum ◽  
Fluorescence Measurements ◽  
Circular Dichroïsm

The size and shape of the Em protein from wheat embryos have been examined by gel filtration, densitometry, ultracentrifugation, and viscosity. Circular dichroism and fluorescence measurements have also been made. Em has an intrinsic sedimentation coefficient, [Formula: see text], of 1.11S and a Stokes radius, RS, of 28.2 Å (1 Å = 0.1 nm) as determined by high performance liquid chromatography gel filtration on a TSK 2000SW column. The partial specific volume [Formula: see text] from density measurements is 0.683 mL/g, a much lower than typical value. The molecular weight from sedimentation equilibrium is 11 200, with no indication for protein aggregation. The intrinsic viscosity [η] of Em is 6.02 mL/g. Circular dichroism shows the molecule to be about 70% random coil. The fluorescence emission spectrum is typical for a tyrosine-containing protein. The hydrodynamic data indicates a poor fit to either a prolate or oblate ellipsoid model; excess hydration or flexibility of the polypeptide chain caused by the rather unusual amino acid composition may be a possible cause. The implications that the low value of [Formula: see text], the high value of RS, and the random-coil configuration of the Em protein may have on its ability to bind water and to contribute to the maintenance of a minimal level of hydration compatible with the sustained viability of the "dry" organism are subjects of an extended discussion. Briefly, it is suggested that Em may provide a matrix of bound water which opposes denaturation of proteins in the "desiccated" cytoplasm of dry plant embryos.

scholarly journals Characterization of proteoglycans from the calcified matrix of bovine bone

1984 ◽  
Vol 224 (1) ◽  
pp. 59-66 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone

The proteoglycans characterized were those isolated from the calcified matrix of mature bovine bone [Franzén & Heinegård (1984) Biochem. J. 224, 47-58]. The average molecular mass of the bone proteoglycan is 74 600 Da, determined by sedimentation-equilibrium centrifugation in 4M-guanidinium chloride. Its sedimentation coefficient (s0(20),w) is 3.04 S. The apparent Mr of its core protein is 46 000, estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chondroitinase ABC-digested proteoglycan. A more likely molecular mass of the core protein is 30 000 Da, as calculated from the molecular mass and the protein content (40%) of the proteoglycan. The bone proteoglycan contains one or probably two chondroitin sulphate chains each with a molecular mass (weight-average) of 33 700 Da and several oligosaccharides both of the N-glycosidically and the O-glycosidically linked type. Antibodies against the homogeneous bone proteoglycans were raised in rabbits. An e.l.i.s.a. (enzyme-linked immunosorbent assay) method was developed that allowed specific quantification of bone proteoglycans at nanogram levels. The specificity of the antibodies was tested by using the e.l.i.s.a. method. The bone proteoglycan showed partial cross-reactivity with the small proteoglycan of cartilage. The antibodies were used to localize immunoreactivity of bone proteoglycans by indirect immunofluorescence in frozen sections of foetal bovine epiphysial growth plate. The fluorescence was entirely found in the primary spongiosa, and no fluorescence was found among the hypertrophied chondrocytes or in the region of provisional calcification.

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

1982 ◽  
Vol 207 (2) ◽  
pp. 297-303 ◽  
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.

Circular dichroism of helical polynucleotide chains

1967 ◽  
Vol 297 (1448) ◽  
pp. 150-162 ◽  
Keyword(s):  
Circular Dichroism ◽  
Nucleic Acids ◽  
Helical Structure ◽  
Optical Rotatory Dispersion ◽  
Random Coil ◽  
Helical Conformation ◽  
Polyadenylic Acid ◽  
V Region ◽  
Circular Dichroïsm ◽  
Circular Dichroic

In contrast to relatively well developed experimental and theoretical studies on polypeptides and proteins (see Gratzer 1967 and McLachlan 1967, this volume) the investigation of optical activity of polynucleotides and nucleic acids were very restricted. The optical rotatory dispersion curves of polynucleotides examined in the visible and near u. v. fit one-term Drude equation regardless of the conformation (Fresco 1961; Levedahl & James 1957; Ts’o, Helmkamp & Sander 1962). Recent circular dichroism (c. d.) measurements of several polynucleotides and nucleic acids (figure 1) indicated clearly the presence of dichroic bands in the u. v. region of base absorption which can be related to the dissymmetrical helical conformation (Brahms 1963). The intensity of circular dichroic bands decreases strongly under the conditions in which the helical structure is unstable and goes to random coil form (Brahms 1964; Brahms & Mommaerts 1964). Thus polyadenylic acid (poly A ) is known according to X-ray data to exist at acid pH in a helical two strand and right handed conformation (Rich, Davies, Crick & Watson 1961). In acid solution the same polyadenylic acid exhibits strong circular dichroic bands which disappear at high temperature (figure 2).

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

1979 ◽  
Vol 183 (2) ◽  
pp. 325-330 ◽  
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.

scholarly journals The C-Terminal Fragment of the Precursor Tail Lysozyme of Bacteriophage T4 Stays as a Structural Component of the Baseplate after Cleavage

1999 ◽  
Vol 181 (9) ◽  
pp. 2739-2744 ◽  
Author(s):  
Shuji Kanamaru ◽  
Nadine C. Gassner ◽  
Nanzhang Ye ◽  
Shigeki Takeda ◽  
Fumio Arisaka
Keyword(s):  
Circular Dichroism ◽  
Structural Component ◽  
Cleavage Site ◽  
Analytical Ultracentrifugation ◽  
Bacteriophage T4 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Precursor Protein ◽  
Terminal Fragment ◽  
Circular Dichroïsm

ABSTRACT Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in β-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.

scholarly journals Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

2010 ◽  
Vol 76 (5) ◽  
pp. 1462-1470 ◽  
Author(s):  
Jes�s Fern�ndez-Lucas ◽  
Carmen Acebal ◽  
Jos� V. Sinisterra ◽  
Miguel Arroyo ◽  
Isabel de la Mata
Keyword(s):  
Circular Dichroism ◽  
Lactobacillus Reuteri ◽  
Equilibrium Analysis ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Differential Scanning Calorimetric ◽  
Calorimetric Studies ◽  
Ph Range ◽  
Α Helix ◽  
Circular Dichroïsm

ABSTRACT A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (Tm ) of 64�C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40�C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.

scholarly journals Physical and chemical properties of human plasma β2-macroglobulin

1978 ◽  
Vol 173 (1) ◽  
pp. 27-38 ◽  
Author(s):  
P K Hall ◽  
R C Roberts
Keyword(s):  
Human Plasma ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.

Purification and characterization of human muscle pyruvate kinase

1977 ◽  
Vol 55 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Richard N. Harkins ◽  
John A. Black ◽  
Marvin B. Rittenberg
Keyword(s):  
Pyruvate Kinase ◽  
Human Erythrocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Psoas Muscle ◽  
Enzyme Inactivation ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Diethylaminoethyl Cellulose

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240 700 and has a sedimentation coefficient (s20,w) of 10.04 S. It contains four subunits with identical molecular weights of 61 000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.

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