A convenient method for the ATPase assay.

A new method for the determination of inorganic phosphorus released in ATPase assay has been evaluated. The method is based on the reduction of a phosphomolybdate complex by Elon in a copper acetate buffer. In contrast to current methods, there is no interference by ATP with color development. There is also less or no interference by other compounds usually present in ATPase assay media. The method is simple, sensitive, and reproducible.     

An improved malachite green assay of phosphate: mechanism and application

The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions.     

An automated assay for Na-K ATPase kinetics

We have developed an automated assay for the Na and K activated ATPase which has been used to determine the enzyme activity in a sample of unknown enzymatic activity or the dependence of the initial rate of reaction on ligand concentration where identical samples of enzyme are used. The interference of nucleotides on the color development of the phosphomolybdate complex has been eliminated by the addition of MgCl₂ to the acid molyb-date solution. Ways of handling the microsomal Na and K stimulated ATPase have been found which insure the stability of the enzyme and facilitate washing through autoanalyzer tubing. Finally, a modification of normal autoanalyzer procedures permits kinetic analysis to be carried out in an automated fashion.     

A colorimetric assay method to measure acetyl-CoA synthetase activity: application to woodchuck model of hepatitis virus-induced hepatocellular carcinoma

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorbance at 580 nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-mul sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-(11)C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-(11)C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. A significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P<0.05). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P<0.05) (total soluble fraction: 876.61+/-34.64 vs. 361.62+/-49.97 mU/g tissue; cytoplasmic fraction: 1122.02+/-112.39 vs. 732.32+/-84.44 mU/g tissue; organelle content: 815.79+/-100.77 vs. 547.91+/-97.05 mU/ g tissue; sedimentable fragment: 251.92+/-51.56 vs. 90.94+/-18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.     

A simple spectrophotometric method for measurement of nematode populations.

A convenient automated method for measuring inorganic phosphate based on the malachite green reaction with a phosphomolybdate complex has been developed. Less than 100 pmol of inorganic phosphate can be readily quantitated by the method which utilizes standard AutoAnalyzer equipment. Inorganic phosphate is measured in sample volumes of less than 0.1 ml and without interference by a number of phosphorylated metabolic intermediates or nucleotides. This methodology is especially useful in the analysis of hydrolytic processes involving phosphorylated substrates.     

Radiochemical methods for the assay of phosphorylase in the direction of glycogenolysis.

Two radiochemical procedures were explored for the determination of phosphorylase activity in the glycogenolytic direction. In the "32P assay method' the formation of labelled glucose 1-phosphate from glycogen and [32P]Pi is measured by the radio-activity that remains soluble after the precipitation of phosphomolybdate with triethylamine. In the "14C assay method' the formation of labelled glucose 1-phosphate from peripherally 14C-labelled glycogen and P1 is determined from the radioactivity that remains soluble after the precipitation of glycogen with ethanol. The 14C assay method requires more preparative work but less circumspection than does the 32P assay method. Both radiochemical methods can be applied where the classical spectrophotometric assay fails. They have the same accuracy and reproducibility, and allow more samples to be handled in parallel. They are not intended for use with crude tissue extracts.     

A simple colorimetric assay method for pyrophosphate in the presence of a 1000-fold excess of orthophosphate: application of the method to the study of pyrophosphate metabolism in mitochondria

A sensitive colorimetric method for the assay of inorganic pyrophosphate with excess of orthophosphate is described. The principle of this method lies in the formation of phosphomolybdate and PPi-molybdate complexes with subsequent extraction of the phosphomolybdate complex by organic solvents and reduction of the PPi-molybdate complex by dithiothreitol and Eikonogen. The sensitivity of the method was from 5 to 120 nmol of PPi in a 2.0-ml sample.     

Characterization of the catalyzed phosphate assay.

A detailed analysis of the catalyzed phosphate assay is presented. This assay uses polyvinylpyrrolidone as a catalyst to form phosphomolybdate complex in a relatively weak acid and hydroxylamine as a reductant to form molybdenum blue. It was found that this assay, which is useful in determining inorganic phosphate in the presence of acid-labile phosphates such as ATP and phosphocreatine, has the following advantages: The assay (a) forms phosphomolybdate at a relatively high pH (pH 1.9–2.1; in some cases even at pH 4.0), (b) is relatively insensitive to interfering reagents, and (c) does not require deproteinization. Conditions are described which make the present assay more sensitive than the Fiske-SubbaRow method.     

A method for preventing sorbitol interference with the determination of inorganic phosphate

A technique is described for preventing interference of sorbitol with the assay of P₁ by modifying the procedure of B. N. Ames (1966, in Methods in Enzymology, E. F. Neufeld and V. Ginsburg, eds., Vol. 8, pp. 115–118, Academic Press, New York). The new method relies on the ability of precipitated protein to bind phosphomolybdate and so allow separation of the P₁ from the soluble sorbitol. The conditions for the formation and precipitation of phosphomolybdate-protein complex and for the subsequent assay of P₁ are described. No unique set of conditions could be found which prevented interference at all sorbitol concentrations tested. Instead, conditions for the elimination of interference by particular sorbitol concentration ranges were established. The application of the procedure to samples containing 0–150 nmol of P₁ and 10–100 μmol of sorbitol is described. Complete recovery of P₁ was achieved after precipitation. Standard plots were linear. Coefficients of variation ranged from 9% with low amounts of P₁ (≤25 nmol) to 2.5% at higher levels (150 nmol). One hundred nanomoles of P₁ gave an absorbance at 700 nm of 0.87. Modifications are described to extend the technique to different sorbitol concentration ranges and other applications of the method are mentioned.     

Mononucleotides of the cell nucleus

1. It has been demonstrated by ion exchange chromatography that the cell nucleus contains mononucleotides of adenine, guanine, cytosine, uracil, together with diphosphopyridine nucleotide, and several uridine diphosphate derivatives; the adenine nucleotides predominating in amount. Nucleotide components in the cell nucleus are in close agreement both quantitatively and qualitatively with those found in the cytoplasm. 2. In calf thymus sucrose nuclei, nucleotide monophosphates can be phosphorylated to the energy-rich triphosphate form without participation of cytoplasmic components. As to the nature of the phosphorylation, it has been shown that there exist certain differences as well as resemblances between nuclei and mitochondria. A distinctive feature of nuclear phosphorylation is that only intranuclear monophosphates seem to be phosphorylated. The process is completely inhibited by cyanide, azide, and dinitrophenol. However, certain reagents which block oxidative phosphorylation of mitochondria, namely dicumarol, Janus green B, methylene blue, and calcium ions, have no effect on phosphorylation within the nucleus. 3. The bulk of mononucleotides is preserved within thymus nuclei after their isolation in sucrose. Nucleotides are surprisingly well retained by nuclei in a sucrose medium whether or not electrolytes are present and in buffers ranging from pH 3 to 10; under all conditions sucrose is required for retention. 4. Dilute acetate in sucrose releases nucleotides from the nucleus below pH 5.1. As to the effective pH of acetate, there is a sharp boundary between pH 5.1 and pH 5.9. At pH 5.9, and above, acetate does not remove nucleotides from the nucleus. The effects of propionate, formate, and monochloroacetate on the nuclei are the same as that of acetate. 5. When nuclei are exposed to a wide variety of conditions a close correlation is found between the retention in the nucleus of nucleotides and of potassium. This suggests that both substances are part of a common complex in the cell nucleus. 6. It has been shown that upon removal of nucleotides and potassium from calf thymus sucrose nuclei by acetate, the ability to incorporate C¹⁴-alanine into nuclear protein is greatly impaired.     

Selective extraction, concentration, and assay of orthophosphate from microliter quantities of cultured mammalian cells

A colorimetric procedure is described for determination of orthophosphate (0.2-2.5 nmol) in sample volumes up to 400 microliters. Orthophosphate is selectively extracted (in the form of phosphomolybdate) into an organic solvent mixture (2-methylpropan-1-ol and petroleum spirit) leaving interfering substances, such as labile organic phosphates, in the aqueous phase. Orthophosphate is then back-extracted into a small volume of aqueous sodium hydroxide. By keeping this volume small, orthophosphate from large dilute samples can be concentrated into small volumes and assayed colorimetrically in microcuvettes using the dye malachite green. The procedure is highly reproducible and insensitive to interfering substances, as shown by comparison with a conventional malachite green assay without the solvent extraction.     

Heparin inhibition of human neutrophil and eosinophil-enriched leukocyte acid beta-glycerophosphatase

Heparin inhibited acid beta-glycerophosphatase (EC 3.1.3.2) from human blood leukocytes, eosinophil-enriched leukocytes, and neutrophils. The inhibition interfered in the hydrolysis of phosphorus from glycerophosphate, not in the formation or detection of colored complexes of phosphomolybdate in the second or color development step in two conventional assays. Heparin inhibited human hypereosinophilic syndrome leukocyte homogenate enzyme activity according to the equation: activity equals 0.946 - 0.087 ln heparin (units/assay) when heparin was varied from 1 to 100 units per assay. At 100 units of heparin per assay, 51% of the original activity remained. Enzyme activity was less in neutrophils than in eosinophils; moreover, the inhibition of neutrophil homogenate by heparin was considerably less than that seen in the eosinophil-enriched leukocyte preparations. In neutrophil homogenates containing 100 units of heparin per assay, 77.1% of activity without heparin was retained. When neutrophil lysates were utilized, less inhibition was observed: e.g., at 1 unit of heparin per assay, 91.7% enzyme activity was retained and at 1000 units, 76.2%; here, activity equals 0.289 - 0.007 ln heparin. The data allowed more precise consideration of the inhibition of acid beta-glycerophosphatase by heparin, and, while confirming quantitatively the greater content of acid beta-glycerophosphatase in eosinophil-enriched leukocyte preparations than in neutrophil preparations, provide experimental support for an acid beta-glycerophosphatase in human eosinophils, which is different from that in human neutrophils. It is more highly susceptible to heparin inhibition than acid beta-glycerophosphatase in human neutrophils from which it is apparently distinct.     

Orthophosphate determination: Interference by mannitol and sorbitol

Mannitol and sorbitol interfered with the orthophosphate estimation based on the formation of phosphomolybdate complex. Binding of molybdate was the cause of interference as increasing the concentration of ammonium molybdate eliminated the interference. Thus, for the estimation of orthophosphate in the presence of mannitol, the concentration of latter must be kept at below inhibitory level.     

Colorimetric estimation of inorganic phosphate in colored and/or turbid biological samples: assay of phosphohydrolases

A simple method of inorganic phosphate determination for colored and/or turbid biological samples is described. The procedure is mild, and so is suitable for routine phosphohydrolase assays. Following deproteinization by ice-cold trichloroacetic (or silicotungstic) acid, the sample was treated with acid-washed charcoal to remove interference due to color. The phosphate in the colorless supernatant was assayed either by measuring the phosphomolybdate spectrophotometrically at 310 nm, following its extraction in organic solvents or by a modified Fiske and Subbarow method. The turbidity interference in the latter case was eliminated either by centrifugation, by sodium dodecyl sulfate treatment, or by extraction of reduced phosphomolybdate blue color by cyclohexanone. Though deproteinization by silicotungstic acid eliminated the turbidity problem, its use in conjunction with charcoal treatment was not convenient.     

Effect of organic anions on the photoreaction of photoactive yellow protein

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.     

The conserved 5' apical hairpin stem loops of bamboo mosaic virus and its satellite RNA contribute to replication competence

Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5' apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C(60) and C(83)) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5' ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.     

An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives

Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.