Purification and properties of ι-carrageenase from a marine bacterium

1984 ◽  
Vol 30 (12) ◽  
pp. 1500-1506 ◽  
Author(s):  
C. W. Greer ◽  
W. Yaphe
Keyword(s):  
Marine Bacterium ◽  
Culture Medium ◽  
Gradient Centrifugation ◽  
Hydrophobic Interaction Chromatography ◽  
Apparent Molecular Weight ◽  
Sucrose Density Gradient ◽  
Resonance Spectroscopy ◽  
Sucrose Density Gradient Centrifugation ◽  
Purification And Properties ◽  
Wall Components

An ι-carrageenase has been purified from the cell-free culture medium of a marine bacterium grown in ι-carrageenan. The enzyme hydrolyzes the β1 → 4 linkages in ι-carrageenan; the major end products, as identified by 13C nuclear magnetic resonance spectroscopy, are ι-neocarratetraose sulfate and ι-neocarrahexaose sulfate. The enzyme was purified by fractionation on Sephacryl S-200 in 2.0 M NaCl and hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. The purified enzyme had an apparent molecular weight of 57 000, and optimum activity was expressed at pH 8.0, 40 °C, in 0.1 M Na+. The enzyme was stable for at least 6 months at 4 °C in 1.0 M NaCl. Removal of the salt, freezing, or lyophilization destroyed enzyme activity. Membrane fractions, prepared by discontinuous sucrose density gradient centrifugation, also exhibited ι-carrageenase activity. Properties of ι-carrageenase suggest an association with cell wall components.

scholarly journals Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

1972 ◽  
Vol 128 (4) ◽  
pp. 817-831 ◽  
Author(s):  
A. Anne Malcolm ◽  
M. G. Shepherd
Keyword(s):  
Molecular Weight ◽  
Thermal Inactivation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Thermophilic Fungus ◽  
Sucrose Density Gradient Centrifugation ◽  
Coenzyme Specificity ◽  
Heat Stable ◽  
Purification And Properties ◽  
Glucose 6 Phosphate Dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000±10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP+- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP+, protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The Km values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3×10−5m-NADP+ and 1.6×10−4m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2×10−5m-NADP+ and 2.5×10−4m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP+ and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme–NADP+–6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ·mol−1 (9.6 and 9.9kcal·mol−1) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5×10−6m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.

Purification and properties of mycoferritin from Mortierella alpina

1972 ◽  
Vol 18 (5) ◽  
pp. 619-622 ◽  
Author(s):  
R. F. Bozarth ◽  
A. Goenaga
Keyword(s):  
High Speed ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Potassium Phosphate ◽  
Sucrose Density Gradient ◽  
Mortierella Alpina ◽  
Protein Subunit ◽  
High Concentration ◽  
Sucrose Density Gradient Centrifugation ◽  
Purification And Properties

A culture of Mortierella alpina Peyroud isolated from soil and grown on Czapek-Dox medium was found to contain a high concentration of mycoferritin (MF). The MF was extracted from lyophilized mycelium by grinding with 0.1 M, pH 7.0, potassium-phosphate buffer and chloroform followed by differential centrifugation. Sucrose density-gradient centrifugation of the concentrated MF resulted in a brownish-yellow band in the region of 60–70 S. Following dialysis and concentration by high-speed centrifugation, the MF was compared to horse-spleen ferritin (HF). The ultraviolet (uv.) and infrared (i.r.) spectra of MF and HF were identical. Negatively stained preparations examined in the electron microscope showed particles of about 10 nm diameter. Sedimentation rates of S20,w = 66 for MF and S20,w = 56 for HF were obtained by analytical ultracentrifugation. The MF preparation contained 83% protein and 17% iron. The molecular weight of the protein subunit was determined by gel electrophoresis to be about 19 300 daltons.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

scholarly journals A glycoprotein associated with the membrane fraction of human B but not T lymphocytes.

1975 ◽  
Vol 142 (6) ◽  
pp. 1416-1424 ◽  
Author(s):  
S Fujita ◽  
S D Litwin ◽  
N Hartman
Keyword(s):  
Membrane Fraction ◽  
Gradient Centrifugation ◽  
Human Lymphocytes ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Sucrose Density Gradient ◽  
Peripheral Blood Lymphocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Lymphoid Lines

A method is described which employs differential centrifugation and sucrose density gradient centrifugation to isolate a membrane fraction from human lymphocytes. Membrane preparations from long-term human cultured B- and T-lymphoid lines, peripheral blood lymphocytes, tonsillar lymphocytes, and thymocytes were analyzed on 0.5% sodium dodecyl sulfate-7.5% polyacrylamide gels stained for protein and carbohydrate. The most important finding was a major glycoprotein of approximately 30,000 daltons associated with the membrane preparations from B lymphocytes. T-lymphocyte preparations did not contain readily detectable amounts of this membrane-associated component. The T-cell lymphoid line MOLT-4 was unique in that it had a narrow protein band at approximately 30,000 daltons which did not contain carbohydrate.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

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