scholarly journals Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome

2021 ◽  
Vol 8 ◽  
Author(s):  
Hideji Yoshida ◽  
Hideki Nakayama ◽  
Yasushi Maki ◽  
Masami Ueta ◽  
Chieko Wada ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gradient Centrifugation ◽  
Essential Amino Acids ◽  
Sucrose Density Gradient ◽  
Bacterial Survival ◽  
Sucrose Density Gradient Centrifugation ◽  
Functional Sites ◽  
Ribosome Binding ◽  
Modulation Factor ◽  
Ribosome Modulation Factor

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

scholarly journals Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

1980 ◽  
Vol 189 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Etsuo Okuno ◽  
Yohsuke Minatogawa ◽  
Masayuki Nakamura ◽  
Naoki Kamoda ◽  
Junko Nakanishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Human Liver ◽  
Analytical Ultracentrifugation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glyoxylate Aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.

scholarly journals Enzyme-mediated binding of tyrosine to brome-mosaic-virus ribonucleic acid

1972 ◽  
Vol 129 (4) ◽  
pp. 969-976 ◽  
Author(s):  
T. C. Hall ◽  
D. S. Shih ◽  
Paul Kaesberg
Keyword(s):  
Amino Acids ◽  
Mosaic Virus ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Alkaline Solutions ◽  
Sucrose Density Gradient Centrifugation ◽  
Virus Rna

Incubation of tyrosine with the RNA from brome-mosaic-virus under conditions favourable for aminoacylation of tRNA resulted in binding of tyrosine to the viral RNA. Sucrose-density-gradient centrifugation showed that tyrosine binds to three and probably to all four of the viral RNA components. The bound amino acid was readily released in mild alkaline solutions. Of 20 amino acids tested, only tyrosine was found to bind. The maximum extent of binding observed was 0.58mol of tyrosine/mol of brome-mosaic-virus RNA.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

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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