scholarly journals Biosynthesis of an insulin precursor by islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 110 (2) ◽  
pp. 281-288 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Progressive Increase ◽  
Exchange Chromatography ◽  
Insulin Precursor ◽  
Single Polypeptide Chain ◽  
Low Concentrations ◽  
Islet Tissue ◽  
Single Polypeptide ◽  
Oxidative Sulphitolysis

1. At 15°, slices of cod islet tissue incorporated [U−14C]proline into proteins soluble in acid–ethanol at a linear rate for 6hr. 2. Initially, all the radioactivity was associated with a polypeptide that had a molecular weight of about 10000 and was appreciably more basic than cod insulin. After 1hr. there was also a significant and progressive increase in the radioactivity of insulin and of fractions intermediate in molecular size and basicity between the polypeptide and insulin. 3. O-Ethyl O-p-nitrophenyl phenylpropylphosphonate markedly decreased the radioactivity both of the intermediate fractions and of insulin, but had no significant effect on the biosynthesis of the polypeptide. In contrast, puromycin inhibited the incorporation of radioactivity into all the fractions. 4. The polypeptide had an activity of less than 0·2 international unit/mg. in the epididymal-fat-pad bioassay. Treatment with low concentrations of trypsin caused a progressive increase in the formation of an insulin-like material, judged by bioassay and ion-exchange chromatography of the digest. 5. Gel filtration of the polypeptide after oxidative sulphitolysis indicated that it was a single polypeptide chain. 6. The results suggest that the polypeptide is an insulin precursor whose formation is inhibited by puromycin and that the steps involved in the conversion of precursor into product are sensitive to O-ethyl O-p-nitrophenyl phenylpropylphosphonate.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals ISOLATION AND PARTIAL CHARACTERIZATION OF THE TWO PRINCIPAL INHERITED GROUP-SPECIFIC COMPONENTS OF HUMAN SERUM

1963 ◽  
Vol 118 (5) ◽  
pp. 711-726 ◽  
Author(s):  
Hartwig Cleve ◽  
John H. Prunier ◽  
Alexander G. Bearn
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Total Amino Acid ◽  
Similar Treatment ◽  
Isolation And Purification ◽  
Single Polypeptide Chain ◽  
Low Carbohydrate ◽  
Single Polypeptide

The group-specific component (Gc), an α2-globulin of human plasma with inherited variations in relative electrophoretic mobility, has been isolated from plasma and partially characterized. The isolation procedure combines ammonium sulfate fractionation, anion exchange chromatography, preparative zone electrophoresis, and gel filtration. The method is suitable for the isolation and purification of the group-specific components. The Gc proteins representing the gene products of the two common homozygous Gc-types) Gc 1-1 and Gc 2-2, have been prepared. Gc belongs to the group of α2-globulins of relatively low molecular weight (4.1S, molecular weight 50,800) and relatively low carbohydrate content (3.3 per cent). The total amino acid composition of the two homozygous group-specific components is very similar; treatment with reducing agents and alkylation provides no evidence for the presence of more than a single polypeptide chain in the Gc molecule.

Xylose metabolism in Pachysolen tannophilus: purification and properties of xylose reductase

1984 ◽  
Vol 30 (11) ◽  
pp. 1330-1336 ◽  
Author(s):  
Günther Ditzelmüller ◽  
Christian P. Kubicek ◽  
Wilfried Wöhrer ◽  
Max Röhr
Keyword(s):  
Xylose Reductase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Xylose Metabolism ◽  
Pachysolen Tannophilus ◽  
Single Polypeptide Chain ◽  
Purification And Properties ◽  
Single Polypeptide ◽  
Reduction Charge

Xylose reductase (xylitol: NADP oxidoreductase, EC 1.1.1.139) has been purified from D-xylose grown cells of the yeast Pachysolen tannophilus by application of DEAE-cellulose ion exchange chromatography, 2′,5′-ADP-Sepharose affinity chromatography, Biogel P200 gel filtration, and dextran blue Sepharose chromatography to approximately 95% homogeneity. It consists of a single polypeptide chain with a relative molecular weight of 35 000–40 000 and an isoelectric point of pH 4.9. The enzyme has a broad substrate specificity similar to that of aldose (or aldehyde) reductases from mammalian tissues. It exhibits Michaelis–Menten type kinetics (Km D-xylose, 162 mM; Km D-xylitol, 212 mM; Km NADPH, 0.059 mM; [Formula: see text], 0.071 mM). The enzyme is specific for NADPH; activity with NADH is below 0.5% of Vmax observed with NADPH. The reduction of xylose is inhibited by NADP, the anabolic reduction charge (NADPH/NADP + NADPH), and also in a complex manner by ATP. At physiological pH values the equilibrium is Keq = 10−10. The importance of these findings for the physiology of xylose fermentation by this yeast is discussed.

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

scholarly journals The purification and some properties of rusticyanin, a blue copper protein involved in iron(II) oxidation from Thiobacillus ferro-oxidans

1978 ◽  
Vol 174 (2) ◽  
pp. 497-502 ◽  
Author(s):  
J C Cox ◽  
D H Boxer
Keyword(s):  
Polypeptide Chain ◽  
Iron Oxidation ◽  
Exchange Chromatography ◽  
Blue Copper Protein ◽  
Blue Copper ◽  
Copper Protein ◽  
Optical Absorbance ◽  
Single Polypeptide Chain ◽  
Arginine Residues ◽  
Single Polypeptide

The ‘blue’ copper-containing protein rusticyanin was purified to homogeneity from cells of the chemolithotrophic bacterium Thiobacillus ferro-oxidans by (NH4)SO4 fractionation and ion-exchange chromatography. The protein, which is stable at low pH, consists of a single polypeptide chain of mol. wt. 16500 and possesses 0.79 (+/- 0.28)g-atom of Cu/mol. The protein, which does not contain arginine residues, has optical absorbance maxima at 287, 450, 597 and 750 nm and is generally similar to azurin. The isolated protein is reduced directly by Fe2+ with a 1:1 stoicheiometry to Cu. On reduction by Fe2+ the absorption peaks at 450, 597 and 750 nm are abolished, with the appearance of a new absorption band at 320 nm. The results obtained are consistent with rusticyanin being the initial acceptor of electrons from Fe2+ during respiratory iron oxidation.

scholarly journals Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain

1987 ◽  
Vol 245 (1) ◽  
pp. 229-234 ◽  
Author(s):  
T Krusius ◽  
V N Reinhold ◽  
R K Margolis ◽  
R U Margolis
Keyword(s):  
Ion Exchange ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Size Fractions ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

Biochemical Characteristics And Inactivation Studies Of An Inhibitor Of Urokinase, From Leucocytes

1981 ◽  
Author(s):  
M Kopitar ◽  
M Drobnič-Košorok ◽  
J Babnik ◽  
V Turk
Keyword(s):  
Cathepsin D ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Inhibitor Molecule ◽  
Active Inhibitor ◽  
Biochemical Characteristics ◽  
Single Polypeptide Chain ◽  
Isolation And Characterization ◽  
Urokinase Inhibitor ◽  
Sepharose 4B

Leucocyte cells are known to contain the acid, thiol and many types of neutral proteinases; among the latter also plasminogen activator. During the last five years appeared also many reports on the endogenous inhibitors of proteinases. In the preceeding publications we reported the isolation and characterization of two inhibitors of proteinases, from leucocytes (of thiol proteinases and of serino proteinases).In the present work we present the biochemical characteristics as well as the inactivation (by cathepsin D) of an urokinase inhibitor isolated from pig blood.Leucocytes were isolated from the peripheral pig blood by dextran procedure. Inhibitor of urokinase could be isolated either from cytoplasm (post granular supernatant) or from the extract of nuclei. Further purification of inhibitor was performed by cation exchange chromatography, by gel filtration on Sephadex G-100 and G-150 and by affinity chromatography on urokinase-Sepharose 4B and on antibody (of urokinase inhibitor )-Sepharose 4B. Inhibitor was isolated in the homogenous form, in a single polypeptide chain with molecular weight of 68 000, designated (leucocyte) Inhibitor-3. This inhibitor has an isoelectric point from 4.4-4.5 and is stable in buffer solutions from pH 3-8, and belongs to the type of so called fast reacting inhibitors. We also established that cathepsin D inactivates this inhibitor by hydrolysis of the inhibitor molecule. The conversion of active inhibitor into inactive protein proceeds catalytically. These last inactivation studies suggest a new pathway by which the active urokinase inhibitor in leucocyte cells may be inactivated.

scholarly journals The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cellulolytic Enzymes ◽  
Botryodiplodia Theobromae ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.

scholarly journals Stability of Arthrobacterd-xylose isomerase to denaturants and heat

1992 ◽  
Vol 285 (3) ◽  
pp. 889-898 ◽  
Author(s):  
M Rangarajan ◽  
B Asboth ◽  
B S Hartley
Keyword(s):  
Gel Filtration ◽  
Xylose Isomerase ◽  
Exchange Chromatography ◽  
Reversible Dissociation ◽  
Low Concentrations ◽  
Competitive Inhibitors ◽  
Optimum Ph ◽  
Relative Stabilization ◽  
Partially Unfolded ◽  
Kinetics Of

There was no inactivation of Mg(2+)-containing Arthrobacter D-xylose isomerase up to 1 h in 0-8 M-urea at 22 degrees C, but over this range there was rapid reversible dissociation into fully active dimers with a midpoint around 4 M-urea, as shown by gradient urea gels with an activity stain, and by ion-exchange chromatography and gel filtration in urea buffers. These dimers must have the A-B* conformation, since the tetramer could dissociate into A-A*, A-B or A-B* dimer conformations, but only residues across the A-B* interface contribute to the active site. The kinetics of inactivation of the Mg(2+)-containing enzyme in 8 M-urea at higher temperatures suggest a partially unfolded Mg-A-B* dimer intermediate with 50% activity, followed by irreversible inactivation coincident with the appearance of unfolded monomer. In 0-4 M guanidinium chloride, a similar reversible dissociation into active dimers occurs, but activity falls, suggesting that A-A* and/or A-B dimers might be part of the mixture. Low concentrations of SDS also give active dimers leading to unfolded monomers, but SDS above 1% (w/v) provides relative stabilization. The apoenzyme is least thermostable (t 1/2 at 80 degrees C, pH 7, = 0.06 h) but Mg2+ stabilizes strongly (t 1/2 = 5.5 h) and Co2+ even more so. Competitive inhibitors or substrates provide a small further stabilization, but this effect is more marked at 80 degrees C, pH 5.5. Together with a marked decrease in optimum pH with temperature, this allows batch isomerizations of glucose under these conditions that produce clean but sweeter syrups.

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