A rapid enzymic procedure for the determination of picomole amounts of UDP-glucuronic acid.

A simple microassay for the determination of UDP-glucuronic acid was developed on the basis of the formation of benzo[a]pyrene 3-glucuronide catalysed by UDP-glucuronyltransferase of guinea-pig liver. As little as 1-5 pmol of UDP-glucuronic acid was detectable in extracts of heat-denatured probes of liver or cultured cells equivalent to 10-50 micrograms of cellular protein.     

Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides

A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.     

The determination of titratable acid and ammonium ions in picomole amounts

A methodological system mainly designed for the use on intratubular urine samples is described, which permits the determination of titratable acid and ammonium ions in samples of a few nanoliters. The pH measurements were performed by means of antimony micro electrodes, the construction of which are described in detail. The hydroxyl ions were added to the samples from a second antimony electrode system, by an electric current. The amount of hydroxyl ions liberated was equivalent to the amount of current used.The ammonium determinations were based upon the fact that hydrogen ions were liberated from the ammonium ions by formaldehyde. The hydrogen ions were titrated in the same manner as the titratable acid.The use of two electrode systems simultaneously inserted in the droplet permitted recordings of the titration curves. The magnitude of methodological errors of these ultramicro methods are the same as those of corresponding methods using milliliter volumes.     

The determination of reduced nicotinamide-adenine dinucleotide and metabolic intermediates in picomole amounts with bacterial luciferase.

Methods that use bacterial luciferase for the assay of NADH in the range from 1 pmol to 1 nmol are described. Optimal conditions for the assay of glycolytic intermediates, tricarboxylic acid-cycle intermediates and related amino acids from milligram amounts of tissue are presented. The whole spectrum of these intermediates can be determined on about 10 mg of liver tissue. The methods are simple, are suitable for routine use, and the instrumentation is inexpensive. The concentrations of glycolytic intermediates in rat livers were determined by conventional spectrometric methods and with luciferase, and the results found to be in good agreement.     

The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Enzymatic methods are described for the analysis of ATP, ATP + ADP, total adenylates, or P-creatine in biological samples. The methods include (i) direct fluorometric procedures for the measurement of 0.1–10 nmol using hexokinase and glucose-6-P-dehydrogenase as the indicator step; (ii) an enzymatic cycling procedure with a sensitivity of 1–50 pmol; and (iii) the measurement of light emission in the luciferin-luciferase system with a sensitivity of 0.1–80 pmol.     

A nonenzymatic method for the determination of picomole amounts of lactate using HPLC: its application to single muscle fibers

A method for the determination of lactate is described in which the esterification of lactate with the uv-absorbing compound alpha-p-dibromoacetophenone is followed by separation and quantitation of the ester by reversed-phase HPLC with detection at 254 nm. The reproducibility, detection limit, and precision of the method are comparable to those of conventional methods which use enzymatic cycling for enhanced performance. The applicability of this rapid and simple method is illustrated with the determination of picomole amounts of lactate in resting and stimulated single muscle fibers.     

A high-performance liquid chromatography method for the analysis of picomole amounts of oligosaccharides

A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintillation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.     

A rapid liquid chromatographic determination of S-adenosylhomocysteine in subgram amounts of tissue.

A new assay is described for the activity of peptidyl-tRNA hydrolase on naturally occurring peptidyl-tRNA molecules. It takes advantage of the differential binding of the tRNA and peptide moieties to DEAE paper.     

Kinetics of the coupled aspartate aminotransferase-malate dehydrogenase reactions and instability of oxaloacetate on anion-exchange resin

The kinetic data of Bryce et al. [C. F. A. Bryce, D. C. Williams, R. A. John, and P. Fasella (1976), Biochem. J., 153, 571–577] indicated anomalous behavior of the coupled aspartate aminotransferase and malate dehydrogenase reactions. From measurements of isotope incorporation (aspartate to malate) and the fact that no enzyme associations could be detected, they concluded that the aminotransferase generates an isomer of oxaloacetate, OAA_a, which is active with the dehydrogenase. In this model, OAA_a would diffuse from the transferase to the dehydrogenase before isomerizing to the equilibrium mixture in which the inactive isomer predominates. (OAA_a was not considered to be either the keto or enol form of oxaloacetate.) We are not able to reproduce the anomalous kinetic or isotope data of these authors. The reasons for the observation of the kinetic anomaly are uncertain. Our isotope experiments, however, indicate that the anion-exchange resin used in this method induces extensive oxaloacetate decomposition making these results unreliable. We also argue that even if there were no experimental errors, the isotope measurements of Bryce et al. would not provide evidence for the oxaloacetate isomer model.     

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.     

Analysis of NAD+ in picomole amounts with sodium [32P]pyrophosphate and NAD-pyrophosphorylase.

A new method is described for the determination of NAD⁺ in picomole amounts. An enzymatic coupling system of NAD-pyrophosphorylase and hexokinase is used to convert sodium [³²P]pyrophosphate and NAD⁺ to [³²P]ADP, glucose 6-[³²P]phosphate, and NMN. The key step in this analysis is the selective adsorption of the reaction product [³²P]ADP, onto activated charcoal with a solution of 1m K₂HPO₄:10% trichloroacetic acid (1:3, v/v, pH 2). The range of concentrations of NAD⁺ that can be measured is 1–200 pmol. The simplicity of the method allows as many as 180 samples to be assayed in 4–5 h. This procedure has been used to quantitate NAD⁺ in crude extracts of germinating wheat embryos.     

Field desorption mass spectrometric measurement of picomole amounts of leucine enkephalin in canine spinal cord tissue

Leucine enkephalin is measured in canine spinal cord tissue in a structurally unambiguous manner. A rapid tissue procurement procedure minimizes enkephalin metabolism. High-performance liquid chromatography purification of brian neuropeptides is followed by field desorption mass spectrometric measurement of leucine enkephalin in spinal cord tissue extracts. Quantification is performed at the 70 ng (126 pmol) g-1 of wet weight tissue, or 70 parts per billion level. The higher homolog of leucine enkephalin, 2ala-leucine enkephalin, is utilized as internal standard. Straight-line statistics are obtained for a series of samples to which a peptide standard is added.     

Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system.

Procedural modifications of the luciferase method for ATP assay in conjunction with enzymatic conversion of AMP and ADP allow the assay of all three adenine nucleotides in quantities ranging from 4 to 20 pmoles. An unmodified Beckman scintillation detector at ambient temperature and in a coincidence mode of operation serves as a suitable instrument for quantitating light emitted by the enzyme preparation. The most significant modifications include use of Ca₃(PO₄) activated crude arsenate extracts of desiccated firefly lanterns, low arsenate concentrations during the assay, and an assay pH of 8.0. Extracts handled in this manner exhibit approximately fivefold higher activity than nonactivated extracts employed at pH 7.4 and 50 mm arsenate. Stability of activated extracts is also somewhat greater than for nonactivated preparations. ADP can be 95% enzymatically converted to ATP by treatment with phosphoenolpyruvate and pyruvate kinase under the conditions described. If myokinase is included, approximately 90% of sample AMP can be converted to ATP. Follwing the appropriate enzymatic treatment, the nucleotides are assayed as ATP and amounts calculated by comparison to curves established for known nucleotide standards. The method is appropriate for perchloric acid extracts of biological tissue and certain considerations necessary for application to experimental situations are described.     

The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

The optimum cofactor requirements for triacylglycerol biosynthesis in rat adipose-tissue homogenates containing mitochondrial, microsomal and cytosolic fractions were investigated. In general the optimum concentrations of cofactors for triacylglycerol biosynthesis were found to differ from those for total fatty acid esterification. The results provided further evidence for the key role of phosphatidate phosphohydrolase in the regulation of triacylglycerol biosynthesis. Albumin was included in the incubation medium to permit the use of concentrations of added fatty acids that would swamp the effects of endogenous fatty acids. The addition of albumin had little effect on the incorporation of palmitic acid and stearic acid into lipids including triacylglycerols. By contrast, a critical concentration of albumin (about 60 muM) was required before incorporation of oleic acid or linoleic acid into triacylglycerols occurred. The system was used to study the incorporation of different 1-14C-labelled fatty acids from a mixture of unesterified fatty acids [palmitic acid 30%; stearic acid 10%; oleic acid 40%; linoleic acid 20% (molar percentages)] separately into the positions 1,2 and 3 of triacyl-sn-glycerols. In general the stereo-specific distribution of the labelled fatty acids incorporated into triacylglycerols paralleled the normal distribution of fatty acids within rat adipose-tissue triacylglycerols, suggesting that the specificities of the relevant acyltrasferases have the major role in determining the positional distribution of fatty acids within triacylglycerols.     

A rapid gas chromatographic-mass spectrometric method for prostaglandin analysis at picomole levels

A rapid method for the qualitative and quantitative determinations of PGE₁, E₂, F_1α, and F_2α is described. Tris-TMS-PGF and mono-TMS-PGB methyl esters are obtained by the reaction of the corresponding PGF and PGE methyl esters with N-trimethylsilylimidazole (TSIM) and piperidine. The reaction is instantaneous, and a single derivative with excellent gas chromatographic properties is obtained for each prostaglandin tested. The presence of very prominent peaks in the mass spectra of the derivatives allows the determination of prostaglandins at picomole levels using multiple ion detection (MID).     

The picomole determination of free and total cholesterol in cells in culture.

An enzymatic, fluorometric method for the determination of free and total cholesterol in cells in culture is presented. The method is simple, requiring one reagent that includes all of the enzymes and a second reagent that increases the pH, which enhances the fluorescence of the product. The method is based on the enzymatic hydrolysis of cholesteryl esters to free cholesterol, the oxidation of cholesterol with the liberation of hydrogen peroxide, and the reaction of this peroxide with a fluorogen to form a fluoresencet product in the presence of a peroxidase. It is rapid, in that free cholesterol can be read in 5 minutes and total cholesterol after 20 minutes. The precision of the method is greater than that obtained from gas-liquid chromatography.     

High-yield isolation of protoplasts from microgram amounts of shoot meristematic tissues and rapid DNA content determination by flow cytometry

This paper describes a novel approach to rapid cell-cycle analysis of shoot meristematic cells. The method involves fixation and disaggregation of meristems into protoplast suspension and flow-cytometric analysis of these protoplasts stained with fluorescent dyes. We have developed a procedure for a high-yield isolation of protoplasts allowing an accurate flow-cytometric analysis with a few micrograms of meristem tissues. We present here determinations of total DNA content of protoplasts stained with propidium iodide in the dicotyledon Sinapis alba, and the monocotyledon Lolium temulentum.