Showing papers in "Methods in Enzymology in 1979"

Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids

Abstract: Publisher Summary This chapter discusses selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods for determining ascorbic acid are numerous. In general, chemical analyses for the vitamin are divided into two groups; the determination of the reduced form and the determination of the oxidized form. The former group of analyses is usually based upon the oxidation–reduction properties of ascorbic acid. These are widely used as the fundamental reactions in the measurement of vitamin C. The latter group of analyses is usually based upon the oxidation of the ascorbic acid and the subsequent formation of a hydrazone or a fluorophore. Best results are obtained if samples, especially plasma, are quickly stabilized with either trichloroacetic acid or metaphosphoric acid and immediately analyzed. Prompt stabilization is especially important in the case of plasma or serum. The greater stability of ascorbic acid in acid solution is because of the decreased tendency for the hydrolysis of the lactone ring with decreasing pH.

The measurement of membrane potential and deltapH in cells, organelles, and vesicles.

Abstract: Publisher Summary The determination of both membrane potential and pH gradient (ΔpH) across a membrane allows the calculation of the proton electrochemical potential difference, or the proton-motive force. The value of the proton-motive force is important for an experimental evaluation of the chemiosmotic hypothesis. It is also an important indicator of the coupling in energy-conversion membrane systems. It is the most sensitive and quantitative indicator of coupling in these systems. As the proton-motive force can be generated by various means, the degree of coupling can be evaluated in systems that lack the complete machinery for oxidative phosphorylation or photophosphorylation. This is in contrast to the more conventional parameters of coupling, such as phosphate/oxygen (P/O), respiratory control, and phosphate potential. The determination of membrane potential or internal pH is not limited to systems of energy conversion; the chapter describes methods for determining the membrane potential in suspensions of cells, organelles, or vesicles.

Properties of detergents.

Abstract: Publisher Summary This chapter reviews the use of detergents for solubilization of membranes and as a solvent medium for membrane proteins. It summarizes the properties of detergents that have been used for these purposes and presents some facts relevant to the choice of detergents for particular experiments. The lipids and proteins of a native membrane interact with each other in a complex fashion that differs in detail from membrane to membrane. The optimal detergent for a particular membrane or membrane protein has to be found empirically and may depend on the type of experiment one wants to do. It should be noted that exchange of one detergent for another is relatively simple. One may use different detergents for initial solubilization and delipidation for subsequent protein characterization and for reconstitution into a lipid vesicle. Integrity of a protein may require contacts among polar groups that are outside the lipid bilayer in the native membrane, possibly including interactions with peripheral proteins adjacent to the membrane or with nucleic acid in the case of a nuclear or viral membrane. Such contacts are influenced by pH, ionic strength, and specific ions.

Plasmids of Escherichia coli as cloning vectors.

Abstract: Publisher Summary This chapter discusses the plasmids of Escherichia coli ( E. coli ) as cloning vectors. The essence of molecular cloning or recombination in vitro is the joining together in vitro of two or more deoxyribonucleic acid (DNA) fragments. One fragment, called the “vector” or “vehicle,” is capable of replication in some host organism and the other(s), referred to as the “cloned or passenger fragment(s),” can be passively replicated when joined to the vector. Hybrid molecules are then put into a host organism by a process called “transformation,” and the clones that contain various arrangements of the joined fragments are isolated. The chapter discusses the procedures that can be used for the generation of DNA fragments and presents the methods for joining the DNA fragments. The chapter discusses the properties of plasmid cloning vectors of E. coli as well as the techniques that can be used to select from a population of bacterial cells the ones carrying recombinant DNA molecules of interest. The elution of DNA fragments from polyacrylamide gels is also discussed in the chapter.

Gel electrophoresis of restriction fragments.

Abstract: Publisher Summary This chapter discusses gel electrophoresis of restriction fragments. There are many designs of an apparatus that can be used for the electrophoresis of deoxyribonucleic acid (DNA) in agarose or polyacrylamide gels. Some of them are very easy to make from glass or perspex, and a simple apparatus can give excellent results. It is often useful to identify a particular sequence in the DNA fragments separated by gel electrophoresis. At present, there are a number of methods available to achieve this purpose. Restriction fragments that bind to a protein may be separated as a complex by filtration through a membrane filter. Radioactive restriction fragments that hybridize to a particular ribonucleic acid (RNA) molecule can be measured by gel electrophoresis after nonhybridized parts of the fragments are digested with the single-strand-specific nuclease S1. Alternatively, nonradioactive restriction fragments, after they are separated by agarose gel electrophoresis, can be probed with radioactive RNA or DNA to mark the positions of specific sequences.

Measurement of growth and viability of cells in culture.

Abstract: Publisher Summary This chapter focuses on cell culture measurements that can be divided into four major categories: (1) visual methods, which employ the use of light microscopy and devices commonly used in hematology; (2) chemical methods, which employ commonly used analytical biochemical procedures adapted to tissue culture; (3) electronic systems using flow-through cells or apertures for measurement of incorporated dyes or cell numbers; and (4) an array of miscellaneous procedures. The handling of cells prior to analysis is extremely important for a variety of reasons: (1) the exogenous growth media may contain components that are being assayed in the cell, (2) leakage of cellular components may occur if cellular integrity is lost at this point, (3) loss of cells may result from severe mechanical handling, and (4) the use of hydrolytic enzymes may lead to the loss of measurable materials.

Plasmid Cloning Vehicles Derived from Plasmids ColE1, F, R6K, and RK2

Abstract: Publisher Summary This chapter discusses the cloning vehicles related to plasmids ColE1, F, R6K, and RK2. Because these replicons have different properties, cloning vehicles derived from one of them may be more suitable for a particular application than vehicles derived from another. Also, because derivatives of a single plasmid do not stably coexist, it is often desirable to have available cloning vehicles from a variety of incompatibility groups to study the interactions among cloned fragments. The ColE1 vehicles pMK20, pMK16, and pMK2004 are very useful for general-purpose cloning because they are maintained in high copy number and provide good selective markers for transformation. Like the ColE1 vehicles, pRK353, a derivative of the antibiotic resistance plasmid R6K, is present in a large number of copies per cell and can be used as a high-copy-number cloning vehicle that can coexist with ColE1. In addition, the R6K replicon has been separated into two components that make up a functional replicon. Plasmid RK2 is unusual because it is maintained in a wide variety of gram-negative bacteria, and therefore RK2 derivatives may be of use as cloning vehicles in gram-negative bacteria other than Escherichia coli ( E. coli ).

[15] Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper

Abstract: Publisher Summary This chapter discusses the process of detection of specific ribonucleic acids (RNAs) or specific fragments of deoxyribonucleic acid (DNA) by fractionation in gels and transfer to diazobenzyloxymethyl (DBM) paper. Small, double-stranded fragments of DNA (30–1800 base pairs) are separated with high resolution on composite gels of agarose and polyacrylamide cross-linked with N,N'-diallyltartardiamide instead of N,N′-methylenebisacrylamide. Following electrophoresis, the cleavage of the crosslinks with periodic acid facilitates the transfer of the denatured fragments to DBM paper. Detection is accomplished with labeled probes. Large fragments of DNA can also be transferred from agarose gels to DBM paper, following partial depurination with dilute acid and strand cleavage with NaOH. The efficiency of transfer is very high and independent of the size of a fragment as covalent linkage allows convenient multiple reuse of the transfers and also allows washings to be done repeatedly under stringent conditions, thereby helping to achieve very low backgrounds. Although many kinds of filter paper can be used, Whatman 540 paper was used in the study described in the chapter because of its excellent mechanical strength and resistance to chemicals. Schleicher and Schuell 589 WH paper also gives good results.

In vitro packaging of lambda and cosmid DNA.

Abstract: Publisher Summary This chapter discusses the in vitro packaging of λ and cosmid deoxyribonucleic acid (DNA). The efficiency of cloning a desired DNA fragment in bacteriophage λ depends on a number of factors such as the availability of a useful λ vector, the probability of obtaining a DNA fragment in a possibly enriched form, the efficiency of conversion of the ligated hybrid DNA molecules to plaque-forming particles, and the availability of a suitable probe for the detection of the hybrid phage. The λ and cosmid DNA, as well as in vitro recombinants, are conveniently packaged using a combination of two lysates, each of which is defective in another step of λ morphogenesis. Empty precursor particles accumulate after induction in bacteria containing a prophage mutant in gene D . This mutation can be complemented by the addition of the missing D protein, most effectively by the supply of an induced λ E – lysogen. This mutation affects the main capsid protein; in its absence, all other head proteins are available in a soluble form. In the presence of adenosine triphosphate (ATP), spermidine, and putrescine, DNA is packaged, the head matures, and tails—components of both lysates—are attached. The result is a DNase-resistant infectious particle, which can be stored like any in vivo -produced phage.

Media and growth requirements.

Abstract: Publisher Summary This chapter reviews culture media and their applications are addressed primarily to the use of cell culture as an experimental tool in fields of research other than analysis of cellular growth requirements. The emphasis is on the types of media available, and on principles involved in selection of the medium that is best suited for the experiment that is to be done. Environmental requirements for cellular multiplication and theoretical aspects of medium development are also reviewed. The chapter provides a brief outline of the types of cultured cells and culture systems. The choice of which culture medium to use and what supplement to add to it is strongly influenced by the type of cell that is to be grown and by the way that cell is to be grown. The chapter discusses the classification of cells: (1) karyotype, (2) multiplication potential, and (3) anchorage dependence.

A centrifuged-column procedure for the measurement of ligand binding by beef heart F1.

Abstract: Publisher Summary This chapter discusses that beef heart mitochondrial ATPase (F1) contains a variety of binding sites for small ligands. These include five sites for adenine nucleotides, two sites for aurovertin, and one or more sites for oxyanions such as 2,4-dinitrophenol and at least one site for Pi. It describes the method suitable for determination of the binding of most of these ligands by the enzyme, utilizes very small amounts of protein (as little as 50/μg), and is highly sensitive because the ligand which was bound to protein is measured in the absence of free ligand. However, the method is not an equilibrium binding procedure, and while it is suited for quantitating the occupancy of binding sites under differing experimental conditions, calculation of dissociation constants from the binding data obtained with F1 or other proteins should be made with caution. The chapter also discusses binding of inorganic phosphate by F1.

Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs.

Abstract: Publisher Summary This chapter describes a general method, which has made possible the sequence analysis of tRNAs from mitochondria, chloroplasts, and a wide variety of prokaryotic and eukaryotic organisms using in some cases as little as 20μg of purified tRNA. The development of in vitro 32 P-labeling techniques has now permitted sequence analysis of nonradioactive tRNAs utilizing adaptations of methods developed by Sanger and co-workers for analysis of uniformly 32 P-labeled RNAs. oligonucleotides with a free 5'-hydroxyl end which are present in digests of nonradioactive nucleic acids can be labeled to high specific activity using T4 polynucleotide kinase and [γ- 32 P]ATP. This approach is used for labeling with 32 P the 5' ends of both mononucleotides present in complete tRNA hydrolysates and oligonucleotides present in T1 or pancreatic RNase digests of tRNA. Using oligonucleotides present in tRNAs of known sequence as standards, the mobility shifts of many commonly occurring modified nucleotides are characterized, which has proved useful in recognizing these modified nucleotides within unknown sequences. The basic steps in the sequence analysis of a nonradioactive tRNA are summarized in the chapter.

[6] The growth of cells in serum-free hormone-supplemented media

Abstract: Publisher Summary This chapter presents the practical details for the growth of cells in serum free hormone supplemented media. In the absence of serum, greater than usual care must be taken in preparation of the synthetic portion of the medium. Careful preparation of water is essential for consistent results with serum-free medium. Serum and even dialyzed serum can mask nutritional requirements of cells in culture. Commercially available powdered media are adequate for serum-free work although each batch must be checked for suitability. Serum also serves the function of a trypsin inhibitor in conventional tissue culture procedures. The serum may be necessary for the repair of trypsinization damage after subculture; or the residual serum left after its removal, even after one or more washes, may be furnishing unknown growth factors.

[6] Preparation of synaptic and nonsynaptic mitochondria from mammalian brain

Abstract: Publisher Summary Preparation of brain mitochondria that are metabolically active and, at the same time, free from nonmitochondrial contaminants, has been a recognized problem. The difficulties encountered in preparing brain mitochondrial populations that fulfill both criteria are inherent in the fact that nervous tissue not only is very heterogeneous but also contains large amounts of lipid and membranous materials. The metabolism of a variety of substances, such as the citric acid cycle intermediates and neurotransmitters, such as glutamate, 4-aminobutyrate, and the catecholamines in the mammalian brain is compartmented. Because the metabolism of these compounds is closely associated with mitochondria and brain mitochondria are heterogeneous in their enzyme contents and complement, appropriate methods have been devised, in which metabolically active and relatively pure brain mitochondrial fractions may be isolated from different functional compartments. The availability of these methods makes it possible to study the control of metabolism in different populations of brain mitochondria and to examine the connection between the heterogeneity of brain mitochondria and metabolic compartmentation observed both in vivo and in vitro.

Long-term culture of dissociated sympathetic neurons.

Abstract: This chapter discusses long-term primary culture of dissociated neurons, a technique that proved to be a valuable tool in studies of neuronal development and function. Such cultures offer the potential for systematic manipulation of the fluid medium surrounding the cells. These neuron-alone cultures are particularly well suited to biochemical analysis as the properties of any function under study are directly attributable to the neurons being cultured, without complicating contamination from other cell types. In dissociated cell culture under appropriate conditions, sympathetic neurons exhibit a normal developmental differentiation and maturation into adrenergic neurons. Medium conditioned by incubation on cultures of appropriate non-neuronal cells, when added to neuron-alone cultures produces an increase in neuronal cholinergic characteristics with a concomitant decrease in adrenergic characteristics. The extracellular environment is critically important in determining the differentiated fate of sympathetically derived neurons.

Clonal strains of hormone-producing pituitary cells.

Abstract: Publisher Summary This chapter discusses clonal strains of pituitary tumor cells that synthesize and secrete prolactin, growth hormone and adrenocorticotropic hormone (ACTH). The rates of biosynthesis of the specific hormonal peptides by these clonal strains are high and they respond in culture to many of the same regulatory factors as normal pituitary cells do in situ. Strains of homogeneous populations of functional cells serve as useful model systems for determining the mechanisms of action of factors that regulate the release and synthesis of prolactin, growth hormone, and ACTH. Several epithelial cell strains have been cloned, and some have been maintained in continuous culture for as long as 10 years without loss of hormone production. Growth hormone (GH) cells produce large amounts of growth hormone and prolactin. The biosynthesis and release of these two protein hormones are modulated by specific factors that have been found to influence the pituitary gland in the intact animal.

[17] Time-resolved fluorescence measurements

Abstract: Nanosecond fluorescence measurements can now be routinely performed and the data can be analyzed in terms of specified decay laws. It is anticipated that nanosecond fluorometry will continue to aid in the elucidation of a variety of excited state mechanistic pathways. This in turn will result in the more sophisticated use of fluorescence probes in biochemistry and cell biology. At the beginning of this decade, Ware9 indicated that the ideal decay fluorometer should permit measurements with samples where the absorbance quantum yield product is 10−10, with high spectral resolution and picosecond time resolution over a wide spectral range. Many of the requirements which seemed so stringent then have now been met. It is likely that the next review on rapid decay techniques will be able to show that picosecond experiments can be carried out with the same degree of confidence that is now possible on the nanosecond time scale.

Transformation and preservation of competent bacterial cells by freezing.

Abstract: Publisher Summary This chapter focuses on transformation and preservation of competent bacterial cells by freezing. In the case of transformation involving a drug resistance marker, the products of transformation may be measured by a pour plate procedure taking advantage of diffusion-limited exposure of cells to the selective drug. The preparation of microbial cultures competent for transformation by added DNA, whether using one of the naturally transformable species or an artificial treatment to render cells permeable to DNA, is typically a multistep procedure occupying at least the greater part of one working day. The preservation of competent or precompetent cultures by freezing has long been exploited in the studies using Bacillus subtilis , Streptococcus pneumoniae , and Haemophilus influenzae .

The avidin-biotin complex in affinity cytochemistry.

Abstract: Publisher Summary This chapter describes the avidin–biotin complex in affinity cytochemistry. The use of affinity methods for the localization, visualization, and subsequent evaluation of specific cellular components is termed “affinity cytochemistry.” In general, the technique is based upon the preparation of a mixed conjugate, comprising a biologically active molecule attached chemically to a potentially perceptible probe, whereby the resultant product retains both detectability and biological activity. A wide spectrum of biologically active molecules is coupled to the above probes, including antibodies. The use of the high-affinity avidin–biotin complex is shown to circumvent some of the problems relating to ferritin–protein conjugation. In addition, this method is employed to unify and facilitate certain aspects of affinity cytochemical techniques. There are numerous steps involved in this step, such as biotin is attached via an appropriate reactive derivative either directly to cell surface functional groups or to a biologically active molecule. The preparation of ferritin–avidin conjugates is described in the chapter.

Measurements of cation transport with metallochromic indicators.

Abstract: Publisher Summary This chapter reviews that sensitive and accurate kinetic measurements of ionized metal ions are central for the understanding of ion transport in biological systems. Among the various cations interacting with biological materials, H + , Ca 2+ , and Mg 2+ have a key role in regulating enzyme activity and cellular functions. Hence, the study of H + , Ca 2+ , and Mg 2+ transport acquires particular significance for the understanding of (1) energy transduction and cellular events such as contraction, secretion, fusion, and excitation and (2) more complex events such as vision and hormone and neurotransmitter action. The chapter also discusses the methods for measuring H + , Ca 2+ , and Mg 2+ , which includes isotope distribution, atomic absorption spectroscopy, specific electrodes, photoluminescent, fluorescence, and absorbance indicators. All of these methods present advantages and disadvantages with respect to selectivity, specificity, time resolution, and sensitivity. It also reviews that metallochromic indicators are substances that undergo color changes when the concentration of free metal ion in the solution changes. The color difference between the free indicator and the indicator-metal ion complex is often very large and can be used for quantitative determination of the concentration of free metal ion in solution.

[59] Inhibitors of the ATP synthetase systems☆

Abstract: Publisher Summary This chapter presents a comprehensive list of compounds that inhibit the synthesis of adenosine triphosphate (ATP) associated with electron transport that is catalyzed by membrane systems present in mitochondria, chloroplasts, and prokaryotic cells. Inhibitors are divided into two groups. Some inhibitors interact directly with the ATPase molecule. In this group, the aurovertins, citreoviridin, Nbf-C1 (4-chloro-7-nitrobenzofurazan), quercetin, tentoxin, and efrapeptin are included. These compounds also react with and inhibit purified soluble ATPase preparations Other inhibitors react with other membrane components of the synthetase complex. These include carbodiimides, oligomycins, venturicidins, organotins, ossamycin, leucinostatin, and Dio-9. Some compounds whose inhibitory effects have not been well characterized are also discussed in the chapter. The chapter discusses the significant physical and chemical properties of these inhibitors.

Elution of DNA from agarose gels after electrophoresis.

Abstract: Publisher Summary This chapter discusses the elution of deoxyribonucleic acid (DNA) from agarose gels after electrophoresis. The studies of genome structure and function rely heavily on the isolation and analysis of the defined DNA fragments. Gel electrophoresis is a simple, high-resolution method of separating specific DNA fragments on the basis of size. Agarose gels at concentrations of 0.1–2.5% resolve DNA from 150–880,000 base pairs, whereas acrylamide gels, ranging from 3–20%, afford a good resolution of fragments in the size range of 10–2,000 base pairs. The chapter describes three new or revised methods for the recovery of DNA from agarose gels. The evaluation is based on the simplicity, speed, yield, and amenability of the purified DNA. The first method involves the electroelution of DNA into slots. The second method involves the electroelution of DNA onto dialysis membranes. The third method describes the process of dissolving gel slices in perchlorate solution.

[6] Improved phosphotriester method for the synthesis of gene fragments

Abstract: Publisher Summary This chapter discusses an experimental study focusing on the improved phosphotriester method for the synthesis of gene fragments During studies with a modified triester approach, it was observed that the pure, fully protected product was difficult to separate quantitatively by the conventional silica gel chromatography from the crude reaction mixture, especially from the components containing several guanine bases It was found that thin-layer chromatography (TLC) on silanized silica gel (RP-2) and KC 18 (RP-18) plates in an acetone–water solvent gave excellent separation of the components containing trityl and hydroxyl groups and also of those differing in sizes The polar component (containing 3′ phosphodiester) generally moved to the solvent front, and the fully protected component (containing the trityl group) was the slowest The mobility of a component containing a 5′-hydroxyl group was between that of the polar and nonpolar compounds The purification of GGCA could not be achieved on silica gel as determined after complete deblocking and TLC on a polyethyleneimine (PEI), whereas purification by TLC on reverse-phase (RP-2) plates gave pure GGCA as analyzed on a PEI plate These results indicated that reverse-phase chromatography of a triester oligonucleotide could yield a pure product at each step of synthesis

Mitochondria from brown adipose tissue: isolation and properties.

Abstract: Publisher Summary Brown adipose tissue (BAT) is heat producing and is specialized for the oxidation of fatty acids No enzymes characteristic of mitochondria in general appear to be missing in BAT mitochondria, but their relative activities are strikingly different These mitochondria have been studied to gain insight into the mechanism of thermogenesis and mitochondrial bioenergetics The rapid proliferation of mitochondria in BAT in connection with birth and cold adaptation renders the tissue suitable for studies of mitochondriogenesis BAT occurs in most neonatal mammals, in certain cold-adapted adult rodents, and in hibernators The state of the tissue and its stage of development vary considerably with the physiological state of the experimental animal; care must be exercised in this respect if comparisons are made between species and between research groups The most commonly used experimental animals are rats, hamsters, and guinea pigs Mitochondrial preparations have been made from ground squirrels, hedgehogs, young lambs, and warm-adapted Syrian hamsters

Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios.

Abstract: Publisher Summary This chapter describes two methods: mechanical cell rupture and cell wall digestion by snail gut juice. It provides an account of new modifications of the two methods, the advantages—greater simplicity and shorter manipulation time. Both, the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, for example, when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods takes only two hours as compared to about six hours for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. The respiratory control ratios are higher for mitochondria obtained by the enzymatic method, although the mechanical method yields similar phosphate/oxygen (P/O) ratios and properly oriented mitochondrial membranes. Representative electron micrographs for the enzymatic and mechanical methods are illustrated in the chapter.

Preparation and analysis of mitochondrial ribosomes.

Abstract: Publisher Summary The chapter discusses the preparation and analysis of mitochondrial ribosomes. Mitochondria contain a distinct species of ribosome that functions in the synthesis of specific polypeptides of the inner mitochondrial membrane. The mitochondrial (mit) ribosomes of microorganisms and higher plants have sedimentation coefficients (s) of 70-80 S. Distinctive features of mit ribosomes include their sensitivity to inhibitors of bacterial protein synthesis, dissociation of monomers at relatively high Mg 2+ concentrations. Mit ribosomes are isolated from many organisms by procedures that include the following steps: (a) isolation of mitochondria, (b) lysis of mitochondria using either deoxycholate or a nonionic detergent, (c) preparation of a mit ribosomal pellet, and (d) separation of monomers or subunits on sucrose gradients. The chapter emphasizes two features: purification of the mitochondria by flotation gradient centrifugation and substitution of Ca 2+ for Mg 2+ during mitochondrial lysis to inhibit nuclease activity. A number of conventional electrophoretic systems are adapted for the analysis of mit ribosomal proteins.