Rapid determination of EUF-extractable nitrogen and boron

Summary A procedure for the rapid determination of EUF-extractable nitrogen (NH₄ ⁺, NO₃ ⁻ and easily soluble organic N compounds) is described. In this procedure the EUF-N fractions are oxidized to NO₃. The oxidation with peroxodisulfate is accelerated by ultraviolet (UV) radiation. This reduces the time of digestion to about 15 minutes. The contents of EUF-extractable N are on the average only between 2–8 mg/100 g soil. Their determination by the new procedure in the form of NO₃ is more precise than the results obtained by digestion according to Kjeldahl. The sum of EUF-extractable N fractions obtained by the new procedure allows to assess the N fertilizer requirements more precisely than is possible when using the EUF-NO₃ fractions alone. Therefore this new procedure constitutes a considerable advantage when working out fertilizer recommendations for agricultural practice.     

A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria

Summary A procedure is described for the calculation of solute fluxes in mitochondria from absorbance measurements. The procedure assumes that mitochondria behave as osmometers and that they are always at osmotic equilibrium.The rates and amounts of K⁺ translocation have been calculated simultaneously, with the photometric procedure and electrometrically, during passive, K⁺ efflux coupled to Ca⁺⁺ uptake and during active K⁺ uptake and passive K⁺ release coupled with anion translocation. Good agreement has been found between the two sets of measurements. The data are compatible with the concept that the energy-linked, ion translocation-coupled, mitochondrial swelling is osmotic in nature. It is concluded that the changes of absorbance are quantitatively related to changes in the inner volume and therefore the photometric procedure can be used to calculate, ion fluxes of osmotically active species under various circumstances.     

Sequential sampling for adult coccinellids in wheat

Adult aphidophagous coccinellids are important predators of cereal aphids in wheat in the Great Plains of the United States for which sampling methods are needed to facilitate improved management. An equation relating the mean number of adult coccinellids per m² in a wheat field to its variance was obtained using Taylor's power law. A sequential sampling procedure was developed to estimate the number of adult coccinellids per m² with constant average statistical precision (standard error/mean). The procedure was constructed by an equation relating the number of adult coccinellids per m² to the number of adult coccinellids per minute of counting incorporating into the Taylor's power law relationship. The procedure involves conducting a series of 1-min counts while walking through a field at a constant velocity of 10 m per minute. After each 1-min count sequential sampling stop-lines are consulted to determine if the specified level of precision has been achieved. Two methods, a statistical procedure and comparison with independent data, were used to assess the consistency with which the specified level of precision was achieved by the procedure. Results indicated that observed precision was close to that specified by the user over a wide range of adult coccinellid density.     

Estimation of DNA, RNA and 125 I- and 3 H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [³H]thymidine and/or [¹²⁵I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine (pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for ¹²⁵I and tritium measurement. Correction for ¹²⁵I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high ${}^3\text{H}{}^{125}\text{I}$ ratios) or by removal of ¹²⁵I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of ¹²⁵I was greater than 99%.     

Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate

A procedure was devised to detect and assay uridine 5′-pyrophosphate (UDP)-glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP-glucuronic acid and ³²P-pyrophosphate, and the ³²P-uridine 5′-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine ³²P-adenosine 5′-triphosphate produced by adenosine 5′-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5′-triphosphate.     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

The continuous bioluminescent assay of ATP has been adapted to the study of Mg²⁺-dependent ATPases, including the (Na⁺,K⁺) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg²⁺-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.     

Preparation of 15N Labeled Nucleosides and Large Scale Synthesis of Labeled Oligonucleotides with a New Type DNA-Synthesizer

Abstract

Microbiological and chemical methods for the preparation of ¹⁵N labeled nucleosides are described. Oligonucleotides are synthesized from the labeled nucleosides on a large scale by the phosphoramidite procedure using a self-developed DNA - Synthesizer. Preliminary ¹⁵N-NMR studies are reported.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (⁶⁴Zn, ⁶⁶Zn, ⁶⁷Zn, ⁶⁸Zn and ⁷⁰Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO₃ and H₂O₂. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO₃ medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique). This full typical procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.     

Reconstitution of an ATP-dependent sodium pump with an ATPase from electric eel and pure phospholipids.

An ATP-dependent sodium pump was reconstituted with an ATPase preparation from the electric eel and purified phospholipids. Vesicles reconstituted by the sonication procedure catalyzed Na⁺ translocation at a rate 10 to 20 times higher than vesicles reconstituted by the cholate-dialysis procedure. A small but reproducible stimulation of Na⁺ transport by valinomycin in the presence of K⁺ was observed.     

Solid-phase synthesis of somatostatin and glucagon-selective analogs in gram quantities

Somatostatin (SS) and two glucagon selective analogs [D -Cys^l4]-SS and [D -Trp⁸, D -Cys¹⁴]-SS have been synthesized in gram quantities by the solid-phase procedure. A general modification of Monahan and Gilon's procedure for esterification of the first protected amino acid onto the chloromethylated resin as well as a general protocol for solid-phase peptide synthesis on Beckman 990 automatic synthesizer are described. A new general procedure for disulfide formation, which involves the adaptation of the “high-dilution” principle to the ferricyanide oxidation and the optimization of the sequence of purification steps as applied to somatostatin and its analogs, yields highly purified peptides (≥ 199% pure) as checked by reverse-phase high-pressure liquid chromatography—which is shown to be a highly sensitive, resolutive, and quantitative analytical tool for evaluation of the homogeneity of peptides.     

Zinc-induced paracrystalline aggregation of glutamine synthetase

The unique capacity of glutamine synthetase to form highly insoluble paracrystalline aggregates in the presence of Zn²⁺ and Mg²⁺ mixtures is the basis of a new simple procedure for the isolation of the enzyme from crude extracts of Escherichia coli. Under optimal conditions (pH 5.85, 25 °C, 1.5 mm ZnSO₄ and 50 MgCl₂ over 95% of the enzyme is precipitated from crude extracts; differential extraction of the precipitate with dilute buffer (pH 7.0) containing 2.5 mm MgCl₂ leads to high yields of almost pure glutamine synthetase. Polyacrylamide gel electrophoresis of the purified enzyme shows it to consist of one major protein and two minor protein components, all of which exhibit glutamine synthetase activity. The major component appears to be identical with the enzyme previously isolated by the older more tedious procedure of Woolfolk et al. (1966). The γ-glutamyl transferase activity of enzyme isolated by the new procedure is the same as that isolated by the older method, but its biosynthetic activity is 25–35% lower. In all other respects examined (i.e., divalent ion specificity, pH optimum, apparent K_m values for substrates, susceptibility to feedback inhibition and physical properties) enzymes prepared by the old and the new procedures are indistinguishable. From studies with pure glutamine synthetase isolated by either procedure, it has been established that paracrystalline aggregation does not occur until 9–10 equivs of Zn²⁺ are bound per mole of enzyme. The high specificity of Zn²⁺ in inducing enzyme aggregation, suggests that its binding provokes a unique conformational state of the enzyme. This is supported by the fact that addition of Zn²⁺ to relaxed (divalent cation free) enzyme elicits a change in the ultraviolet spectrum of the enzyme that is qualitatively different from that caused by either Mg²⁺ or Mn²⁺. Moreover, in contrast to Mg²⁺, the binding of Zn²⁺ decreases the fluorescence associated with the binding of 2-p-toludinyl-naphthalene-6-sulfonic acid to the enzyme, suggesting that Zn²⁺ binding is accompanied by a decrease in the number of exposed hydrophobic regions on the enzyme.     

Overcoming the Problem of Non-Ideal Liquid Ion Exchanger Selectivity in Microelectrode Ion Flux Measurements

Ion-selective microelectrodes are a powerful tool in studies on various aspects of cell membrane biology in both animal and plant tissues. Further application of this technique is, however, limited to a large extent by the problem of non-ideal selectivity of the liquid ion exchanger used in the preparation of microelectrodes for ion flux measurements. Because of this problem, which is persistent in many commercial liquid ion exchangers, the microelectrode does not discriminate between the ion of interest and other interfering ions (for example, Mg²⁺ and Ca²⁺; Na⁺ and K⁺), thereby leading to inaccurate concentration readings and, consequently, inaccurate flux calculations. In this work we show that the existing analytical procedure to overcome this problem, using the inverted Nicolsky-Eisenman equation, is inadequate, and suggest an alternative analytical procedure that can be applied directly to the data obtained with commercially available liquid ion exchangers. We show that this alternative procedure allows accurate measurement of ionic concentrations with non-ideal ion-selective microelectrodes in the presence of interfering ions, and illustrate the method by direct experiment using Ca²⁺ and Mg²⁺ as a “case study”. Several more examples are given, further illustrating practical applications of the method for study of plant responses to salinity, osmotic and reactive oxygen species stresses.     

Separation of alpha and beta chains of rabbit globin using SDS-polyacrylamide gel electrophoresis

A procedure to separate the α and β globin chains of rabbit hemoglobin, denatured with sodium dodecyl sulfate in the presence of mercaptoethanol, on a column of polyacrylamide gel was developed. The identity of the two separated chains was verified by (a) differences in distribution of radioactivity between the chains when the hemoglobin samples were labeled uniformly with various ³H- or ¹⁴C-labeled amino acids; (b) the analysis of the chain distribution of radioactivity in purified hemoglobin isolated from rabbit reticulocytes, pulse-labeled with [³H] leucine; and (c) the separation pattern of a mixture of authentic [α-³H]- and [β-¹⁴C]-labeled globin chains. The globin chains of human hemoglobin A also could be separated in a similar manner. This procedure is particularly useful when only microgram quantities of hemoglobin are available for study.     

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA^fMet in solution. The procedure involves conversion of cytosine residues to N⁴-[¹⁴C]methylcytosines by treatment with ¹⁴CH₃NH₂ and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces ¹⁴C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA^fMet has been found to contain only six reactive cytosines: C₁, C₁₆, C₁₇, C₃₅, C₇₅ and C₇₆. In addition, slow reaction at C^m₃₃ was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA^fMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.     

ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl₂ method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na⁺ + K⁺-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b₅. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na⁺ + K⁺-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.     

Solid-state and solution conformation of scleroglucan

A potentiometric-titration procedure, in which samples are always exposed to an excess of I₂-KI has been developed for measuring iodine-binding capacity of starches. Binding capacity of amylose under these conditions is ～30% as opposed to 20% by conventional potentiometric titration. Spectrophotometric absorbance is essentially the same for either method, but is proportional to potentiometric values only in the excess-iodine titration procedure. Effects of variation of concentration of I₂, KI, and phosphate buffer and of temperature on the reaction have been examined. Calculations based on concentration of reactants in solution indicate that the binding species varies from I₃^? at 10^?1m KI to I₁₁^? at 5 × 10^?4m KI.