Incorporation Of Radioactive Phosphate Research Articles

Cyclic amp-stimulated phosphorylation of a high molecular weight endogenous protein substrate in sub-cellular fractions of molluscan nervous system

Cyclic AMP stimulated the incorporation of radioactive phosphate from ATP into endogenous phosphoproteins in a 20,000 × g supernatant, as well as in a 150,000 × g supernatant, prepared from Helix nervous system homogenates. No effect of cyclic AMP was observed in the respective pellet fractions. A part (but not all) of the cyclic AMP-stimulated activity in both supernatants was due to phosphorylation of an endogenous protein substrate, of apparent molecular weight 120,000 daltons as determined from its mobility in polyacrylamide gels. Little phosphorylation of 120,000 dalton material was observed in the particulate fractions, both in the presence and absence of cyclic AMP. When the 20,000 × g pellet was incubated with radioactive ATP and an exogenous calf ovary protein kinase, cyclic AMP stimulated the phosphorylation of the 120,000 dalton material. The results suggest that both membranes and cytosol from Helix nervous system contain 120,000 dalton substrates for cyclic AMP-dependent protein kinase. However, no kinase catalyzing phosphorylation of these substrates is recovered in membrane fractions following homogenization of the tissue.

The synergetic enzyme theory of muscular contraction: II Relation between Hill's equations and functions of two-headed myosin

Based on the assumption of nonidentical two heads of myosin it is pointed out that a strong motive force is generated in actomyosin pair only when ATP-decomposition occurs co-operatively at the both heads and that the tension-independent part of shortening heat is liberated when an ATP molecule is decomposed only at the burst head. These two actions of actomyosin pair are related to the two states of force-generator in Huxley-Simmons' model. Elementary cycles at different positions in a sarcomere are interacted each other through feedback loop via sliding motion of muscular filaments. Due to this synergetic interaction the rate constant for the rate-determining step of elementary cycle has a dependence on velocity v of shortening such as k = k ° + κv. From these functions and properties of actomyosin system in vivo, the following properties of muscle are explained consistently in a quantitative manner: (1) Hill's equation on the relationship between tension and velocity of shortening, (2) damped oscillations in tension and in muscular length around steady state, (3) Hill's energy equation improved in 1964, (4) the chemical equivalence of shortening heat, (5) the influence of tension on the incorporation of radio-active phosphate into ATP and (6) the asymmetric activation by actomyosin system only for the forward reaction, the decomposition of ATP.

Effect of factors causing excitation and inribition on polyphosphoinositide metabolism of the rat liver

The rate of incorporation of radioactive phosphate into the polyphosphoinositide (PPI) fraction is about 10 times greater than into other phospholipids (PL) of the normal rat liver. Rapid postmortem changes in the PPI content in the liver were found. During exposure to the various factors used, the only PL fraction in which changes were found was the PPI fraction. Changes in the content and also in the intensity of metabolism of PPI in the liver were similar in direction during exposure to these factors to those in the brain. The PPI fractions are the most labile PL fractions in the liver.

Phosphorylation of Proteins from the Brains of Mice Subjected to Short-term Behavioral Experiences

This chapter describes the phosphorylation of proteins obtained from enriched synaptosomal fractions from the brains of mice subjected to short training experiences. An outline of the experiment is presented by a diagram in the chapter. One of a pair of 6-8-week-old male mice was injected intracranially with approximately 20 μCi [ 32 P] orthophosphate in 20 μl water, while the other was injected with 20 μCi of carrier-free [ 33 P] orthophosphate from ICN, Irvine, Calif., U.S.A. Twenty-nine minutes after injection one mouse was placed in a jump-box training apparatus and trained. The untrained mouse stayed in the home cage during the entire 45 min and referred as Quiet. At the end of the training, both mice were decapitated after minimal ether anesthetization. The brains were removed and the olfactory bulbs and cerebellum excised. Proteins of synaptosome-enriched fractions of mouse brain exhibited an increase in the incorporation of radioactive phosphate during 15 min of avoidance conditioning. The increase was initiated between 5 and 10 min of the training and could be detected 30 min beyond the training period.

Brain phospholipid metabolism during ischemia.

Molecular mechanisms accounting for the vulnerability of brain to ischemic damage are incompletely understood. To assess the role of membrane integrity, phospholipid metabolism is investigated in subcellular organelles of brain following varying degrees of ischemia. Global brain ischemia is produced in rabbits by combining hypotension and hypoxia (4% oxygen). Restoration of blood pressure and oxygen after a three-minute isoelectric EEG results in full recovery, while a five-minute isoelectric EEG results in either a lack of recovery or neurological deficits. Quantitative analysis of seven phospholipids in subcellular organelles shows no consistent pattern of phospholipid changes following ischemia. Incorporation of radioactive phosphate, on the other hand, discloses moderate alterations following five-minute ischemia. These findings indicate that brain ischemia provokes phospholipid changes, but their role in impairing membrane integrity and in initiating irreversible ischemic brain damage is uncertain.

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells.

Phospholipid metabolism in alveolar macrophages of lungs. (32P)orthophosphate incorporation in pulmonary surfactant phospholipids in rat in vivo and in vitro (author's transl)

Phospholipid Metabolism in Alveolar Macrophages of Lungs [32P]Orthophosphate Incorporation in Pulmonary Surfactant Phospholipids in Rat in vivo and in vitroPulmonary surfactant phospholipids play an important role in the surface tension activity of the lung.Incorporation of [32P]orthophosphate into rat‐lung pulmonary‐surfactant phospholipids was studied in vitro and in vivo after separation on silicic‐acid‐column chromatography and by one and two‐dimensional thin‐layer chromatography. While they represent a small part of the phospholipids of the whole lung, pulmonary‐surfactant phospholipids show a rapid turnover, particularly in vitro.After in vitro incubation, phosphatidylcholine and lysophosphatidylcholine show very important specific activities; by contrast, phosphatidylethanolamine and sphingomyelin have a very low turnover. Thus phosphatidyldimethylmethanolamine‐methylation pathway, demonstrated in the lung, is not very important.Phosphatidylglycerol is labeled more markedly in vivo than in vitro indicating, for this lipid, a possible transfer from lung tissue to pulmonary‐surfactant cells. In diphosphatidylglycerol specific activities are always very low.The incorporation of radioactive phosphate into phosphatidylinositol, more important in vitro than in vivo, is probably related to the phagocytosis activity of these cells.Our work demonstrates the existence of a phospholipid biosynthesis into pulmonary surfactant but cannot explain the metabolic relations between lung tissue and macrophagic alveolar cells investigated as a possibility in our results.

Apparent Average Length of the Transcriptional Unit in Bacteria

The kinetics of radioactive phosphate incorporation into the adenosine and guanosine nucleoside triphosphate termini of bacterial ribonucleic acid (RNA) was studied. Knowledge obtained in a previous investigation of the kinetics of phosphate incorporation into their precursors, adenosine 5'-triphosphate and guanosine 5'-triphosphate, allowed calculation of the average half-lives of these termini, which were found to be approximately 170 s at 21.5 C for both. The ratio between the number of nucleotides incorporated into the interior of RNA chains per second and the number of termini synthesized per second was calculated by several methods and found to be between 4,000 and 8,000. Assuming that the initiation of synthesis of a RNA chain by deoxyribonucleic acid-dependent RNA polymerase always produces a triphosphate termini and that some termini do not have half-lives so short as to not be seen in this study (less than 10 s), this is the apparent average length of the transcriptional unit. The implication of these findings to the genetic organization of transfer RNA genes is discussed.

Brain phosphoproteins: the effect of various behaviors and reminding experiences on the incorporation of radioactive phosphate into nuclear proteins

An increase of radioactive phosphate incorporation into nonhistone acid-extractable protein occurs in the brain nuclei of naive mice or rats during 5 min of active one-way avoidance training. The increase is not triggered by motor activity, handling, or the experimental conditioning stimuli. An increase does occur, however, in the brains of previously trained animals when they perform the avoidance, and it occurs in rats trained on previous days when they are simply handled or placed in the training apparatus. This “reminder effect” does not occur in rats that have not been trained, but have repeatedly received only unavoidable footshock in the apparatus.

Activation by adenosine 3':5'-monophosphate of a membrane-bound phosphoprotein phosphatase from toad bladder.

Adenosine 3':5'-monophosphate (cyclic AMP) caused a decrease in the net rate of incorporation of radioactive phosphate into a specific protein (protein D) in a membrane fraction from toad bladder. Moreover, when the membrane protein was prelabeled with radioactive phosphate, cyclic AMP caused an increase in the net rate of removal of radioactive phosphate from this specific protein. Certain agents were shown to be selective inhibitors of membrane-bound protein D kinase or protein D phosphatase. With the help of these agents, it was concluded that cyclic AMP caused the activation of membrane-bound protein D phosphatase. The present data, together with earlier studies, are compatible with the possibility that the cyclic AMP-induced activation of a membrane-bound phosphoprotein phosphatase in toad bladder, with the consequent dephosphorylation of protein D, may be responsible for the physiological effects of antidiuretic hormone on sodium and/or water transport in this tissue.

Rapidly labelled RNA in heterotrophically cultured tissues of Nicotiana tabacum

The RNA metabolism in tissue cultures of Nicotiana tabacum has been studied by incorporation of radioactive phosphate. The RNA was extracted by phenol-kresol-hydroxychinon and fractionated by MAK columns. A rapidly labelled RNA fraction was eluted from the MAK column after the rRNA components. Its base ratio differed from the latter in that the AMP content (28,6%) was higher than the GMP content (26,0%). Sedimentation analysis on sucrose gradients showed a sedimentation coefficient of 37 -38s. The life time of the 37-38s RNA fraction in chase experiments was 3,5-4 hours. Differential centrifugation of tissue homogenates revealed that the labelled 37-38s RNA was associated with particles sedimented by low speed centrifugation. No 37-38s RNA could be detected in the ribosomal fraction and the supernatant. During chase experiments label appeared in the rRNA components. It could be excluded by thin layer chromatographic separation of 32P labelled compounds that this label originated from 32P-o-phosphate or labelled organic phosphates except of the rapidly labelled 37-38s RNA. From this it was concluded that the 37-38s RNA is a precursor of the rRNA.

Phosphorylation of Endogenous Protein of Rat Brain by Cyclic Adenosine 3′,5′-Monophosphate-dependent Protein Kinase

Abstract The ability of proteins in various subcellular fractions of the rat cerebrum to act as substrates for a partially purified cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase has been studied in vitro. Distribution of substrates generally paralleled the distribution of activity of protein kinase as well as of adenylate cyclase and cyclic nucleotide phosphodiesterase. Although all particulate subfractions were capable of becoming phosphorylated, highest substrate activities were found in the synaptic plasma membrane, microsomal, and synaptic vesicle fractions. Substrates in the microsomal fraction included both membrane and ribosomal proteins. The synaptic plasma membrane and the microsomal fractions exhibited a high endogenous rate of incorporation of radioactive phosphate from [γ-32P]ATP into phosphoprotein, compared to other subcellular fractions, and this rate was increased by addition of protein kinase and cyclic AMP. Phosphorylation of the synaptic plasma membrane fractions indicated that some membrane proteins were at least comparable to and possibly better than the histones in effectiveness as substrate for the enzyme. The results support other evidence implicating the cyclic AMP system in the molecular events underlying synaptic transmission in the nervous system.

Incorporation of 32P into ribosomal RNA, transfer RNA and inositol hexaphosphate in germinating pea cotyledons

1. 1. RNA synthesis in germinating pea cotyledons has been studied by incorporation of radioactive phosphate. 2. 2. Sucrose density gradient centrifugation of RNA extracts showed that 28-S and 18-S rRNA are synthesized in an approximately linear fashion for up to 6 days. Radioactive label is also incorporated into tRNA. 3. 3. RNA extracts also contained highly labelled low molecular weight material which obscures the tRNA labelling region on sucrose gradients. The labelled material is soluble in 5 % trichloroacetic acid and can therefore be resolved from tRNA. 4. 4. The trichloroacetic acid-soluble material from RNA extracts was labelled very rapidly with 32P on germination and its labelling approximated to curves for water uptake up to 36 h. This was resolved by DEAE-cellulose chromatography and Sephadex G-25 chromatography into two major components, one of which was impure, being associated with carbohydrate and peptide material. In some preparations the partially purified 32P-labelled material co-chromatographed with ATP and in others with inorganic phosphate; it is likely that the latter case represented breakdown of ATP. The other rapidly labelled compound was identified as inositol hexaphosphate. 5. 5. 32P-labelled inositol hexaphosphate was found only in germinating cotyledons and was not detected in the developing embryo tissue.

5'-Deoxy-5'-fluoro-thymidine, a biochemical analogue of thymidine-5'-monophosphate selectively inhibiting DNA synthesis.

5′‐Deoxy‐5′‐fluorothymidine (5′‐fluorothymidine) inhibits the DNA synthesis of cells of the Ehrlich ascites carcinoma of the mouse (carcinoma) in vitro, as estimated by the incorporation of radioactive phosphate. About 90 or 50% inhibition is obtained at concentrations of 1 mM or 70 μM, respectively. The synthesis of neither RNA nor protein is inhibited by 5′‐fluorothymidine. The inhibition of DNA synthesis cannot be reversed by thymidine. In cell‐free extracts obtained from carcinoma cells, 5′‐fluorothymidine strongly inhibits the phosphorylation of thymidine‐5′‐monophosphate, but hardly at all that of thymidine. A large accumulation of thymidine‐5′‐monophosphate is found among the phosphorylation products derived from thymidine in the presence of 5′‐fluorothymidine. Thus, 5′‐fluorothymidine acts as inhibitory analogue of thymidine‐5′‐monophosphate in the thymidine‐5′‐monophosphate kinase reaction. The inhibition of thymidine‐5′‐monophosphate kinase is competitive. The affinity of the analogue for the enzyme is about three times higher than that of the substrate. The enzyme is also inhibited by thymidine, but its inhibitory activity is only one tenth that of 5′‐fluorothymidine. 5′‐fluorothymidine provides protection of the enzyme against inactivation brought about by incubation in the absence of the substrate. 5′‐fluorothymidine is degraded by pyrimidine phosphorylases at a rate of only one sixth that of thymidine. 5′‐fluorothymidine inhibits the multiplication of carcinoma cells in vitro either completely or by 60% at concentrations of 100 or 10 μM, respectively. The increase in fermentation, normally associated with this cell multiplication, is much less influenced by 5′‐fluorothymidine. Thus, the effect of 5′‐fluorothymidine is primarily seen in cell division, but to a less extent in the development of cytoplasmic processes (similar to unbalanced growth). Since 5′‐fluorothymidine cannot be phosphorylated at C‐5′ it must be assumed to act unchanged as such. This is in contrast to the classical base, or nucleoside, analogues, which, for inhibitory activity, have to be transferred intracellularly into the nucleotides (lethal synthesis). The independence of 5′‐fluorothymidine of lethal synthesis should be of advantage with regard to resistance problems for a possible use of this compound in cancer chemotherapy.

Oxidative phosphorylation in yeast II. An oxidative phosphorylation-deficient mutant

1. Mitochondria from the mutant Saccharomyces cerevisiae DH 1 oxidized members of the tricarboxylic acid cycle, d(−)- and l(+)-lactates, ethanol, NADH, and N, N, N′, N′-tetramethyl- p-phenylenediamine with P/O ratios found in manometric experiments to be as low as 0.1 to 0.4. The low phosphorylation efficiency was not improved by a large excess of ADP or glucose + hexokinase, by serum albumin, or by the supernatant from an homogenate of non-mutant yeast. The weak phosphorylation was suppressed by oligomycin. In short-term polarographic experiments, higher P/O ratios, determined by a radioactive phosphate incorporation technique, were consistently found. 2. The oxidation activity of the mutant mitochondria was the same in the presence and absence of hexokinase + glucose and was not raised by ADP. 3. 2,4-Dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone at uncoupling concentrations stimulated the oxidation of all substrates with the exception of l(+)-lactate. This indicates that the coupling mechanism was not abolished in the mutant mitochondria. In media of about 0.15 osmolarity, Mg 2+ was required for dinitrophenol to stimulate the oxidation. 4. ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of mutant mitochondria displayed two pH optima like that from non-mutated yeast. However, the oligomycinsensitive component with pH optimum at pH 9.5 was substantially reduced in the mutant mitochondria. 5. Cytochrome a content was found to be lower in the mutant mitochondria. 6. Possible changes in the oxidative phosphorylation system of the mutant mitochondria are discussed.

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.

Quantitative Microdetermination Of Enzymes in Sweat Gland

ONE OF THE manifestations of cystic fibrosis is failure to produce a normal hypotonicity in the sweat. This defect may be due to inability to retrieve and chloride from a precursor fluid. It may thus be failure of a sodium mechanism for secreting from the sweat lumen into the extracellular fluid. Such a pump mechanism has been investigated in the gland of the albatross and other marine birds. 1 It has also been investigated in the electrolyte transport across erythrocyte membranes. 2 The present study was undertaken to test the possibility that failure of this mechanism was involved in the defect of the sweat glands in cystic fibrosis. The mechanism in the avian salt glands and the erythrocyte membrane has been shown to be accompanied by a turnover of phospholipids as indicated by incorporation of radioactive phosphate, particularly into phosphatidic acid. This phosphatidic acid

The incorporation of radioactive phosphate into the ribonucleic acid of cell-free preparations of Azotobacter vinelandii

1. 1. A cell-free preparation from Azotobacter vinelandii is capable of incorporating [ 32 P]P i into RNA in the presence of Mg 2+ ions and 3-phosphoglyceric acid (or phosphoenol pyruvate). 2. 2. The 105 000 × g particulate fraction as well as the 105 000 × g supernatant fraction are essential for the incorporation. 3. 3. The incorporation is considerably stimulated by the addition of AMP, ADP, ATP or Poly-A at a concentration of 1·10 −3 M. Higher concentrations usually lead to inhibition. 4. 4. The possibility of the labelling of RNA by the freely reversible reaction catalysed by polynucleotide phosphorylase has been ruled out. 5. 5. The radioactive RNA isolated from the incubation mixture behaves as s-RNA and all of its four nucleotidic components are radioactive.

The incorporation of radioactive phosphate into the ribonucleic acid of cell-free preparations of Azotobacter vinelandii

1. A cell-free preparation from Azotobacter vinelandii is capable of incorporating [32P]Pi into RNA in the presence of Mg2+ ions and 3-phosphoglyceric acid (or phosphoenol pyruvate). 2. The 105 000 × g particulate fraction as well as the 105 000 × g supernatant fraction are essential for the incorporation. 3. The incorporation is considerably stimulated by the addition of AMP, ADP, ATP or Poly-A at a concentration of 1·10−3 M. Higher concentrations usually lead to inhibition. 4. The possibility of the labelling of RNA by the freely reversible reaction catalysed by polynucleotide phosphorylase has been ruled out. 5. The radioactive RNA isolated from the incubation mixture behaves as s-RNA and all of its four nucleotidic components are radioactive.

The incorporation of radioactive phosphate into the ribonucleic acid of cell-free preparations of Azotobacter vinelandii

The incorporation of radioactive phosphate into the ribonucleic acid of cell-free preparations of Azotobacter vinelandii