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1.
Orthophosphate is rapidly transported into cultured cells and subsequently incorporated into numerous compounds. A high-performance liquid chromatographic method that enables the measurement of 32Pi incorporation into acid-soluble metabolites in cultured cells treated with exogenous 32Pi is described. Baseline resolution and quantitative recovery of 12 ribonucleotides are accomplished in less than 75 min. In cultured, beating rat heart cells, the concentration and extent of labeling by 32Pi of most phosphorylated metabolites were unchanged in cells treated with the anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). The method is generally applicable to the investigation of phosphate transport and incorporation by numerous cell types under various experimental conditions. 相似文献
2.
Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein 总被引:2,自引:0,他引:2
Y Kagawa L W Johnson E Racker 《Biochemical and biophysical research communications》1973,50(2):245-251
Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid32Pi-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little32Pi-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [32P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation. 相似文献
3.
Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of 32Pi into phosphatidylinositol (PI) in rat diaphragm incubated in Ca2+-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of 32Pi into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Caa+-chelating properties; the inhibition of the incorporation of 32Pi could be completely reversed by the addition of excess Ca2+ but not Mg2+. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca2+, rather than extracellular Ca2+, was involved in the effect. In contrast to its inhibition of the incorporation of 32Pi EGTA enhanced the incorporation of [3H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [3Hlinositol by the tissue. Our results suggest that the level of intracellular Ca2+ plays a role in the regulation of the incorporation of 32Pi into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of 32Pi into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of 32Pi into α-glycerophosphate. 相似文献
4.
Maguire JJ Kirby HR Mead EJ Kuc RE d'Anglemont de Tassigny X Colledge WH Davenport AP 《PloS one》2011,6(11):e27601
Kisspeptins, the ligands of the kisspeptin receptor known for its roles in reproduction and cancer, are also vasoconstrictor peptides in atherosclerosis-prone human aorta and coronary artery. The aim of this study was to further investigate the cardiovascular localisation and function of the kisspeptins and their receptor in human compared to rat and mouse heart. Immunohistochemistry and radioligand binding techniques were employed to investigate kisspeptin receptor localisation, density and pharmacological characteristics in cardiac tissues from all three species. Radioimmunoassay was used to detect kisspeptin peptide levels in human normal heart and to identify any pathological changes in myocardium from patients transplanted for cardiomyopathy or ischaemic heart disease. The cardiac function of kisspeptin receptor was studied in isolated human, rat and mouse paced atria, with a role for the receptor confirmed using mice with targeted disruption of Kiss1r. The data demonstrated that kisspeptin receptor-like immunoreactivity localised to endothelial and smooth muscle cells of intramyocardial blood vessels and to myocytes in human and rodent tissue. [125I]KP-14 bound saturably, with subnanomolar affinity to human and rodent myocardium (KD = 0.12 nM, human; KD = 0.44 nM, rat). Positive inotropic effects of kisspeptin were observed in rat, human and mouse. No response was observed in mice with targeted disruption of Kiss1r. In human heart a decrease in cardiac kisspeptin level was detected in ischaemic heart disease. Kisspeptin and its receptor are expressed in the human, rat and mouse heart and kisspeptins possess potent positive inotropic activity. The cardiovascular actions of the kisspeptins may contribute to the role of these peptides in pregnancy but the consequences of receptor activation must be considered if kisspeptin receptor agonists are developed for use in the treatment of reproductive disorders or cancer. 相似文献
5.
(i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 mm, whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly. 相似文献
6.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated. 相似文献
7.
Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na+-dependent inorganic phosphate (Pi) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na+-dependent 32Pi cotransport in cultured fetal rat cortical neurons, where >90% of saturable Pi uptake is Na+-dependent. Saturable, Na+-dependent 32Pi uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na+-dependent Pi transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent Km values for Pi and Na+ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (>60%) Na+-dependent Pi uptake, with a threshold for maximal Pi import of 1–2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na+-dependent Pi import rates. These data establish the presence of a Na+-dependent Pi cotransport system in neurons of the CNS, demonstrate the Ca2+-dependent nature of 32Pi uptake, and suggest that the neuronal Na+-dependent Pi cotransporter may import Pi required for the production of high-energy compounds vital to neuronal metabolism. 相似文献
8.
Glucagon and insulin both stimulated the 32P-labelling of ribosomal protein S6 in rat hepatocytes that had been incubated with 32Pi. Glucagon selectively enhanced the labelling of the tryptic peptide phosphorylated by cyclic AMP-dependent protein kinase, demonstrating that 6 S is a physiological substrate for this enzyme. Insulin stimulated the phosphorylation of distinct tryptic peptides, at least one of which appears to be very close in the primary structure to the sites phosphorylated by cyclic AMP-dependent protein kinase. 相似文献
9.
1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with 32Pi reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of Pi. (ii) 32Pi, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [32P]ADP when 32P-labelled mitochondria are incubated with non-radioactive Pi.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of Pi; one readily communicates with external Pi and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external Pi and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase. 相似文献
10.
N. Dupont-Mairesse P. Galand 《Biochimica et Biophysica Acta (BBA)/General Subjects》1975,404(2):214-220
We have studied the phosphorylation of soluble proteins from uterine extracts by an endogenous protein kinase. The analysis of phosphorylation patterns by polyacrylamide gel electrophoresis did not reveal any significant difference in this respect between the soluble proteins from control of 17-β-estradiol stimulated uteri. In both cases, three main components with mol. wt of about 120 000, 60 000 and 45 000 appear preferentially phosphorylated.Estrogen-induced protein did not coincide with any phosphorylated component, although some migrated very closely to it. This was observed whether phosphorylation was performed on uterine extract incubated with [γ-32P]ATP or on intact organs incubated in the presence of 32Pi.We conclude that whatever the role of estrogen-induced protein, it is unlikely to be subjected to regulation through the phosphorylation process. 相似文献
11.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated. 相似文献
12.
We previously demonstrated inhibition of Na+-dependent 32Pi transport in canine renal brush-border membranes in association with NAD+-induced ADP ribosylation of membrane protein(s) and postulated that NAD+ inhibits Pi transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD+ resulted in release of Pi which was prevented by EDTA. It was proposed that NAD+-mediated inhibition of 32Pi transport might occur through this mechanism. To determine whether NAD+ inhibited 32Pi transport by a mechanism other than or in addition to release of Pi, we compared Na+-dependent 32Pi counterflow in brush-border membrane equilibrated with Pi or with Pi generated from NAD+. Release of Pi from NAD+ incubated with brush-border membrane was confirmed. The increased uptake of 32Pi which was demonstrated in brush-border membrane equilibrated with Pi was not measured when intravesicular Pi was generated from a concentration of NAD+ which effected ADP-ribosylation of brush border membranes (100 μM NAD+). In contrast, increased uptake of 32Pi was demonstrated when intravesicular Pi was generated from 1 μM NAD+ which did not effect ADP ribosylation. Mg2+-dependent ADP ribosylation of brush-border membrane incubated with NAD+ was demonstrated which persisted during the time interval of 32Pi uptake measurements. Our findings are compatible with the hypothesis that NAD+-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of Pi transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg2+ and decreased ADP ribosylation. 相似文献
13.
The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-32P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation. 相似文献
14.
The possibility of a mobile carrier model for phosphate transport in rat liver mitochondria was examined on the basis of counterflux experiments. The rate of Pi uptake and Pi exchange were identical and depended on the external pH value. The alkalization of the suspending medium of Pi-preloaded mitochondria induced an efflux of Pi. The induction of a net Pi efflux by alkalization of the external medium stimulated the rate of 32Pi uptake. Arsenate was shown to be an alternative substrate for Pi-carrier. A net efflux of inorganic arsenate (Asi), induced by alkalization of the external medium, also supported an acceleration of the 32Pi uptake. When mitochondria were first preloaded with Asi and 32Pi and then diluted into a more alkaline buffer free of Asi, a transient uptake of 32Pi was observed. These results are discussed in terms of reorientation of the active site of the Pi carrier under the conditions where a net efflux of Pi or Asi occurred. This conclusion was supported by a change in the accessibility of the SH groups of the carrier toward poorly permeant thiol reagents during that process. 相似文献
15.
31P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the Pi signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/Pi ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/Pi ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/Pi ratio was observed. Intracellular pH can be calculated from determination of the Pi-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases. 相似文献
16.
K. Hotta 《Journal of cellular biochemistry》1975,3(4):333-337
Myosin catalyzed exchange between 32Pi and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of 32Pi in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange 32Pi and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of 32Pi incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested. 相似文献
17.
Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi ATP exchange reaction with bound Pi The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers. 相似文献
18.
B I Kanner R Serrano M A Kandrach E Racker 《Biochemical and biophysical research communications》1976,69(4):1050-1056
Coupling factor 6 (F6) and mitochondrial ATPase inhibitor were isolated from the rutamycin-sensitive ATPase complex of bovine heart mitochondria by heating and fractionation with ethanol. F6 appeared in acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and urea as a single band corresponding to a molecular weight of 8,000. This protein which is required for the 32Pi-ATP exchange in submitochondrial particles treated with silicotungstate was very sensitive to trypsin. 相似文献
19.
Mrunal S. Chapekar Michael Bustin Robert I. Glazer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,838(3):351-354
The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [32P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with 32Pi, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [γ-32P]ATP. 相似文献
20.
In view of the importance of Pi in the control of cell metabolism, it was of interest to study the mechanism and regulation of Pi uptake by ascites tumor cells. For this purpose, the incorporation of 32Pi into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease 32Pi uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 Mr anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases 32Pi incorporation at concentrations as low as 2 × 10?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na+ + K+)-ATPase and its dephosphorylation, stimulates the rate of incorporation of 32Pi into cells and also raises the net inorganic phosphate level. The stimulation of 32Pi incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K+, which is known to stimulate (Na+ + K+)-ATPase and its dephosphorylation, decreases 32Pi incorporation. These observations suggest that anion transport and energy-dependent Na+ and K+ movements may be closely coupled to the intact cell. 相似文献