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PubMed1999

Crystallization and preliminary X-ray diffraction studies of perchloric acid soluble protein (PSP) from rat liver.

Perchloric acid soluble protein purified from the cytosol fraction of rat liver has been crystallized in a form suitable for high-resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in cross-sectional diameter were produced by the hanging-drop method using polyethylene glycol (Mr = 8 kDa) as precipitant. These crystals diffract to 2.44 A on an in-house X-ray source and to 1.8 A using a bending-magnet beamline at ESRF Grenoble. The crystals belong to the cubic space group P213 with a = 89.90 A and two molecules per asymmetric unit, as indicated from a Vm value of 2.12 A3 Da-1 and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound and xenon that bind to the protein.

Animals↗
PubMed1991

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution.

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.

Adenosine Triphosphate↗
PubMed1991

Phosphorus 31 magnetic resonance spectroscopy of human placenta and quantitation with perchloric acid extracts.

Phosphorus 31 magnetic resonance spectroscopic studies of fresh placental tissue are reported that indicate resonances for adenosine triphosphate, inorganic phosphate, sugar phosphates-phosphomonoesters, and phosphodiesters. Perchloric acid extract methods were used to further characterize and quantitate phosphorous metabolites in term human placentas by phosphorus 31 magnetic resonance spectroscopy. The perchloric acid extracts give enhanced resolution of phosphorus signals and allow identification of other phosphorus metabolites including small amounts of phosphocreatine. Emphasis was placed on quantitating adenosine triphosphate levels in the acid extracts with the use of the external reference standard hexachlorocyclotriphosphazene in a coaxial capillary system. Adenosine triphosphate levels measured in this way ranged from 0.404 to 0.709 mumol per gram wet weight. Comparison with an internal standard method with phosphocreatine is also reported. Contribution to the measured high-energy phosphate pool from blood in the highly vascularized tissue was found to be relatively large and could range from 30% to 50% of the total adenosine triphosphate measured.

2,3-Diphosphoglycerate↗
PubMed1978

Kinetic studies on the formation of nitrosamines I. Formation of dimethylnitrosamine in aqeous solution of perchloric acid.

The kinetics of nitrosation of dimethylamine (DMA) in aqueous perchloric acid solution have been studied using a differential spectrophotometric technique. The rate law is Initial rate = e[DMA]0 [nitrite]2 0 [H+]/(f + [H+])2 where [DMA]0 and [nitrite]0 represent initial stoichiometric concentrations. At 310.0 K and mu = 2.0 M, e = (2.2 +/- 0.2) X 10(-5) M-1 s-1 and f = (1.28 +/- 0.02) X 10(-3) M. The associated activation energy is 56 +/- 3 kJ mol-1. A clear inhibition of the nitrosation rate by ionic strength has been observed in which only the kinetic parameter (f) has an effective change. It is concluded that under the experimental conditions of this work only the dinitrogen trioxid is the effective carrier for the nitrosation.

Dimethylnitrosamine↗
PubMedDate not supplied

[Muscle glycoproteins (soluble in perchloric acid) after denervation and drug stimulation].

Muscle proteins soluble in perchloric acid contain about 75% of carbohydrates presented by neutral hexoses, fucose and hexuronic acids. Three days and two weeks after denervation the content of the glycoproteids protein part is unchanged, a month later it increases by 116%. The content of carbohydrate component following denervation lowers considerably and a month later is on the average 40% higher than in the muscle of healthy rats. Administration of muscular RNA restores the content of the protein component only by the end of a month; administration of pyrogenal normalizes the content of both protein and carbohydrate components.

Animals↗
PubMed1995

A critical assessment of brain metabolites: analysis of perchloric acid extracts using proton nuclear magnetic resonance.

A critical assessment of perchloric acid (PCA) brain tissue extracts for precise identification and quantitation of brain metabolites using in vitro proton nuclear magnetic resonance (1H NMR) spectroscopy was studied. One pulse with a presaturation NMR experiment was used. The chemical shifts and coupling networks of the major brain metabolites as a function of pH were characterized by using individual model compounds and a model mixture solution. We found that the conditions of the PCA solution are essential for accurate interpretation of NMR spectra of brain metabolites. The maximum spectral resolution was obtained at pH 4.92. Caution is necessary when using high resolution 1H NMR spectroscopy to identify and quantify brain metabolites.

Animals↗
PubMed1996

Isolation of perchloric acid soluble, heat stable, ethanol extractable protein from Mycobacterium tuberculosis.

The perchloric acid soluble, heat stable, and ethanol insoluble antigen of M. tuberculosis (TB-PBE) was prepared, and antigenicity of this antigen was studied in vivo and in vitro. TB-PBE showed a single band of 60 kDa by SDS-PAGE. Sera from the patients with active pulmonary tuberculosis did not react with this antigen by ELISA. A delayed hypersensitivity skin reaction was induced with this antigen and was correlated with the reaction with PPD. Skin biopsy was performed in this skin lesion induced by TB-PBE and stained by H-E and immunohistochemical methods. TB-PBE induced an inflammatory lesion similar to a lesion induced by PPD. Blastogenic activity of the peripheral blood mononuclear cells stimulated by TB-PBE increased, and showed a peak reaction at 7 days after stimulation. The blastogenic activity changed in a dose-dependent manner. After stimulation with TB-PBE, mononuclear cells were analyzed by FACS. DR+ T cells and CD4/CD8 ratio increased after stimulation by TB-PBE. These cells secreted IL-2, not IL-4 after stimulation with TB-PBE. In the immunofluorescence test, mouse antiserum against TB-PBE showed a positive reaction with M. tuberculosis and showed cross-reactivity with M. bovis and other atypical mycobacteria, but not with S. aureus. With these results, it is evident that TB-PBE is an antigen which can induce cell mediated immunity in vivo and in vitro.

Animals↗
PubMed1993

Glutathione, but not glutamine, is detected in 13C-NMR spectra of perchloric acid extracts from C6 glioma cells.

Glutamine, which is expected to be produced by C6 glioma cells, is not detected in both amino-acid analyses and 13C-NMR spectra of perchloric acid extracts of cells incubated for 4 h with [1-13C]glucose in the absence of extracellular glutamine. However, the resonances of a glutamate-linked product are observed in these spectra. The analysis of the pH dependence of chemical shifts from various glutamate-derived compounds shows that the observed resonances came from glutathione. Glutamine and glutathione signals are in close proximity on the frequency scale, leading to possible misinterpretation of the spectra.

Brain Neoplasms↗
PubMed1978

Buffer-exchange column for rapid separation of carcinoembryonic antigen from perchloric acid.

A Sephadex G-50 medium (Pharmacia) buffer-exchange column has been developed for rapidly changing the medium for carcinoembryonic antigen from perchloric acid to acetate buffer. Analytical recovery of the extracted antigen exceeded 95%. Plasma for analysis can be so prepared and samples ready for analysis within an hour. The technique also results in uniform assay volume, thus eliminating this variable from the assay.

Buffers↗
PubMed1988

The effect of perchloric acid fixation on DNA preservation in the salivary gland of Bradysia hygida.

In Bradysia hygida (Diptera, Sciaridae) well spread polytene chromosomes, free of cytoplasm, and with good morphology are consistently obtained if before squashing in acetic acid solution, the salivary glands are fixed in 7% perchloric acid containing small amounts of ferric ions (SAUAIA et al. 1971). We show here that, with regard to the preservation of total incorporated 3H-thymidine and with regard to the relative autoradiographic labelling of expanded chromosome regions as compared to the labelling of a non-expanded one, this method is equivalent to fixing the salivary glands in ethanol-acetic (3:1). We show also that if this kind of preparation is subject to mild acid hydrolysis a small amount of the total 3H-labelled material may be lost.

Animals↗
PubMedDate not supplied

Regional distribution of perchloric acid soluble proteins during the development of rat brain.

The regional contents of perchloric acid (PCA)-soluble proteins were determined during the development of rat brain. The soluble proteins in cortex, mesencephalon and medulla oblongata decreased gradually to adult age, whereas the levels in cerebellum increased significantly 12 days after birth followed by a decline to adult age. The developmental changes in the soluble proteins in retina, which showed the highest content of these proteins, were different from that of the other regions. Changes in the staining intensities of the main protein bands following SDS-gel electrophoresis were observed during the development of all regions. The patterns of the soluble proteins from retina and cerebellum were less complex than those of the other brain regions and liver and resembled that of the chromosomal high-mobility group proteins and H 1 histones.

Aging↗
PubMed1993

Analysis of perchloric-acid-soluble Mycobacterium bovis bacille bilié de Calmette-Guérin antigen.

The perchloric-acid-soluble Bacille bilié de Calmette-Guérin (BCG) antigen (BCG-P) was investigated for the diagnosis of tuberculosis. Monoclonal antibodies against BCG-P were produced. After SDS-PAGE, silver staining and immunoblotting were done. Pathological specimens of pulmonary tuberculosis, BCG organism itself and atypical mycobacterium were stained using the indirect immunofluorescent technique with the strongest one among these monoclonal antibodies. Intradermal skin test and ELISA were also done with BCG-P. BCG-P was composed of a major 65-kD protein and a minor 61-kD protein. Fourteen monoclonal antibodies were produced which were all IgM. These antibodies reacted with the 65-kD protein which also showed cross-reactivity with Mycobacterium tuberculosis in pathologic tissues and atypical mycobacterium. BCG-P did not react with the sera of patients with active pulmonary tuberculosis. However, intradermal skin test with BCG-P showed positive reactions in the patients with a positive purified protein derivative of tuberculin skin test. This antigen could be a good antigen to evaluate the success of BCG vaccination.

Antibodies, Monoclonal↗
PubMed1976

Loss of ATP in micromolar amounts after perchloric acid treatment.

Treatment of fluid samples containing known amounts of ATP with 6.0 N perchloric acid (PCA) results in a total loss of 65-71% when the initial concentrations of ATP ranged between 0.5 to 50 muM. Half of this loss was attributed to desensitization of firefly extract (luciferin-luciferase reaction) while the remaining loss was presumably due to adsorption of ATP to perchlorate precipitate upon neutralization. Similar treatment of solutions with higher initial concentrations (100-1000 muM) resulted in apparent total losses averaging 22%. These losses were due solely to desensitization of firefly extract by neutralized PCA. Both the adsorption and desensitization phenomena must be taken into account when the ATP content is measured from tissue extracts and fluid samples subjected to this procedure.

Adenosine Triphosphate↗