Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

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.     

Measurement of phenylalanine hydroxylating activity in vivo

A gas chromatographic-mass spectrometric method was developed for analyzing phenylalanine and tyrosine (Tyr) in plasma and brain. With this procedure, we were able to show that¹⁸O₂ is incorporated into Tyr in vivo and that the presence of¹⁸O-Tyr in plasma is a relative measure of phenylalanine hydroxylating activity. Treatment withp-chlorophenylalanine decreases the¹⁸O incorporation into Tyr. Because of the simplicity of the procedure and its easy adaptability to human studies, the incorporation of¹⁸O₂ into endogenous constituents might serve as a useful diagnostic procedure for some metabolic disorders, such as phenylketonuria. These results also indicate that labeling with¹⁸O cannot be used to measure the turnover rate of brain catecholamines as previously proposed.     

Phosphorylation assay in liquid scintillation counter using C̆erenkov radiation of P: Application to photophosphorylation

A simple procedure is described for the assay of phosphorylation using C?erenkov radiation to detect ³²P in a liquid scintillation counter. Unreacted ³²P_i is first removed from the reaction mixture as the phosphomolybdate complex by butanol/benzene extraction. Addition of ammonium hydroxide to the remaining aqueous fraction avoids color quenching, phase separation, and instability in the counting rate during measurement of ³²P. Application of this procedure to several photophosphorylation systems is included.     

A simple method for synthesizing [γ-32P]nucleoside triphosphates using [32P]acetylphosphate and acetate kinase

A simple, rapid, and inexpensive method is described for the synthesis of γ-³²P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [³²P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less.     

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.     

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 × 50 μm². Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN^?, P^?, S^?. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P^? content. The CN^?, P^?, and S^? ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.     

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.     

Can ultrasonic energy efficiently speed 18O‐labeling of proteins?

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the ¹⁸O‐decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the ¹⁸O‐isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

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⁺.     

The release of endonuclease I from Escherichia coli by a new cold shock procedure

A new cold shock procedure has been developed for releasing large quantities of endonuclease I from $\text{E. coli}$, which neither involves EDTA-lysozyme treatment nor osmotic shock. Treatment of cells with ice-cold 0.1M Tris-0.2M KCl buffer, pH 7.4 results in the release of endonuclease I into the medium. Although the loss of endonuclease I from the cells is a rapid process, its recovery in the shock fluid is gradual and approaches maximum in about 90 minutes. Certain divalent metal ions such as Mg⁺⁺ and Mn⁺⁺ strongly inhibit the release of endonuclease I. The cold shock procedure is rather selective and the mechanism of the release of endonuclease I is different from that of osmotic shock procedure.     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

Sequential individual resonance assignments in the 1H-nmr spectra of polypeptides and proteins

Recently, a new procedure for the assignment of protein ¹H-nmr spectra was introduced that relies on stereochemical considerations of proton–proton distances in polypeptides and on the use of two-dimensional nmr for obtaining ¹H-¹H through-bond and through-space connectivity maps. In the present paper a particular aspect of this assignment procedure is discussed in more detail, i.e., how to obtain individual resonance assignments from identification of amino acid side-chain spin systems and identification of neighboring residues in the amino acid sequence.     

Analysis of mRNA 5'-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography

A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m⁷GpppN′) or cap 1 (m⁷GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′-O-methylnucleosides (N″m) of cap 2 (m⁷GpppN′mpN″m) and internal N⁶-methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl-³H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure.     

Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [¹⁴C]δ-aminolevulinic acid with a high yield of conversion (?60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (?30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [¹⁴C]protoporphyrin preparation.     

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%.     

Biosynthesis of steroidal withanolides in Withania somnifera

Administration of 24-methylene-cholesterol-[28-³H] to Withania somnifera, yielded [³H] radioactivity in the isolated withaferin A and withanolide D, whereas administered 24-(R,S)-methyl-cholesterol-[28-³H] was not incorporated into these compounds. 24-Methylene-cholesterol is, therefore, proposed as a sterol precursor of the withanolides. A novel procedure is described for the isolation of withanolides from W. somnifera. This method in conjunction with an improved procedure for administration of labelled sterols and mevalonolactone produces a greatly increased yield of labelled withanolides.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

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.