Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation

1980 ◽  
Vol 58 (9) ◽  
pp. 677-682 ◽  
Author(s):  
Trevor Lukey ◽  
Floyd F. Snyder
Keyword(s):  
Density Gradient ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Adenosine Kinase ◽  
The Other ◽  
Deoxycytidine Kinase ◽  
Deoxyguanosine Kinase ◽  
Assay Conditions ◽  
Kinase A ◽  
Broad Specificity

The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 μM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 μM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 μM deoxyguanosine was inhibited approximately 90% by 10 μM deoxycytidine or dCTP and was inhibited 70% by 200 μM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 μM, phorphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 μM, was partially inhibited by 200 μM adenosine, 34%; 200 μM deoxyguanosine, 10%; and 100 μM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 μM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.

scholarly journals Ribonucleic Acid Biosynthesis in Human Leukocytes

Blood ◽  
1966 ◽  
Vol 28 (2) ◽  
pp. 188-200 ◽  
Author(s):  
MARTIN J. CLINE
Keyword(s):  
Density Gradient ◽  
Ribonucleic Acid ◽  
Turnover Rate ◽  
Rna Synthesis ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Acid Biosynthesis ◽  
Total Load ◽  
Human Leukocytes ◽  
Particle Ingestion

Abstract Phagocytosis has profound effects on several aspects of the RNA metabolism of human leukocytes. The major changes induced by particle ingestion appear to be (1) an increased uptake of pyrimidine precursors from the suspending medium, (2) a contraction in the size of the nucleotide pool, (3) an accelerated rate of destruction of preexisting RNA, and (4) an increased rate of RNA synthesis. Sucrose density gradient analysis of the newly synthesized RNA suggests that several classes of RNA are involved in this process. The increased turnover rate of the nucleotide pool and of the cellular RNA of the leukocyte is proportional, within limits, to the total load of ingested particles.

ANALYSIS OF RIBOSOMES AND POLYSOMES DERIVED FROM RABBIT MAMMARY GLANDS DURING PREGNANCY AND LACTATION

1971 ◽  
Vol 49 (4) ◽  
pp. 667-NP ◽  
Author(s):  
I. D. HERRIMAN ◽  
G. D. BAIRD ◽  
JUDY M. BRUCE
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Gradient Analysis ◽  
Late Pregnancy ◽  
Sucrose Density Gradient ◽  
Mammary Glands ◽  
Pregnancy And Lactation

SUMMARY Whole-ribosome and polysome-enriched fractions were prepared from the mammary glands of rabbits during late pregnancy and lactation. The composition of the fractions was determined by sucrose density gradient analysis and electron microscopy. The range of size of polysomal aggregates was similar in the late-pregnant and lactating gland, with aggregates containing five to nine ribosomal units predominating. However, the amount of polysomes relative to monosomes was invariably found to increase after parturition. The greater portion of this increase was accounted for by the increased abundance of aggregates containing five to nine units.

scholarly journals STUDIES ON SEEDS

1971 ◽  
Vol 48 (3) ◽  
pp. 533-541 ◽  
Author(s):  
Hilton H. Mollenhauer ◽  
Clara Totten
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Morphological Changes ◽  
Gradient Centrifugation ◽  
Lipid Vesicles ◽  
Sucrose Density Gradient ◽  
The Other ◽  
Animal Tissues ◽  
Sucrose Density Gradient Centrifugation ◽  
Simple Lipid

Two structurally distinct lipid vesicles are present in pea and bean cotyledons during the first few days of germination. Both were isolated by sucrose density gradient centrifugation without significant morphological changes. Lipid vesicles of one type were elongated into a sausage-like or flattened-saccular shape, and were interassociated into sheets which were usually one vesicle thick. These sheets remained intact during homogenization and centrifugation, because some of the lipid vesicles in the sheet were interconnected through their bounding membranes, and because there seemed to be a bonding substance between adjacent vesicles. These vesicles were called "composite" lipid vesicles to distinguish them from the more usual, or "simple," lipid vesicles of other plant and animal tissues. Lipid vesicles of the other type were usually larger than the composite lipid vesicles and were always spherical in form. These vesicles remained single and did not interassociate into sheets. They were probably equivalent to the simple lipid vesicles of other tissues.

scholarly journals Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

1969 ◽  
Vol 115 (5) ◽  
pp. 1063-1069 ◽  
Author(s):  
T. Scott-Burden ◽  
A. O. Hawtrey
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Lithium Chloride ◽  
Ribosomal Rna ◽  
Gradient Analysis ◽  
Extraction Procedure ◽  
Liver Microsomes ◽  
Sucrose Density Gradient ◽  
Rat Liver Microsomes ◽  
Phenol Extraction

1. Treatment of washed rat liver microsomes in a medium containing 0·12m-sucrose, 12·5mm-potassium chloride, 2·5mm-magnesium chloride and 25mm-tris–hydrochloric acid buffer, pH7·6, with 2m-lithium chloride at 5° for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting 14C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH–2,6-dichlorophenol-indophenol reductase, NADH–neo-tetrazolium reductase, NADH–cytochrome c reductase and ribonuclease activities. 5. 3H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of 3H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.

Sucrose Density Gradient Analysis of 1,25-Dihydroxyvitamin D3Binding in Human Breast Tumors*

1983 ◽  
Vol 56 (4) ◽  
pp. 686-691 ◽  
Author(s):  
Sylvia Christakos ◽  
Alan Sori ◽  
Stuart M. Greenstein ◽  
Thomas F. Murphy
Keyword(s):  
Density Gradient ◽  
Gradient Analysis ◽  
Breast Tumors ◽  
Sucrose Density Gradient ◽  
Human Breast

A Factor Capable of Dissociating Rat Liver Ribosomes

1971 ◽  
Vol 49 (12) ◽  
pp. 1301-1306 ◽  
Author(s):  
G. Ross Lawford ◽  
Jutta Kaiser ◽  
W. C. Hey
Keyword(s):  
Ionic Strength ◽  
Ammonium Sulfate ◽  
Density Gradient ◽  
Rat Liver ◽  
Messenger Rna ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Small Subunit ◽  
Temperature Dependent ◽  
Liver Ribosomes

A factor capable of dissociating rat liver monomeric ribosomes into 60 S and 40 S subunits has been partially purified and characterized.The factor was prepared by extracting a fraction of rat liver enriched in its content of native subunits with 0.05 M triethanolamine–HCl, 1.0 M KCl, 0.01 M MgSO4, and 2 mM dithiothreitol. The activity of the preparation was assayed by testing its ability to dissociate monomeric ribosomes into subunits which were detected by sucrose density gradient analysis. The ribosomes used as substrate were prepared by dissociating polysomes in the presence of puromycin, 0.5 M KCl, and 3 mM MgSO4 and subsequently reassociating the subunits into monomers by lowering the ionic strength. The factor acts only on ribosomes freed of both messenger RNA and nascent protein by associating with the small subunit. The activity was time and temperature dependent, reaching a plateau after 30 min at 30 °C.The factor has been partially purified by ammonium sulfate fractionation between 35% and 65% saturation and by treatment at 40 °C for 15 min to precipitate ribosome-aggregating substances.

Sign in / Sign up

Export Citation Format

Share Document