Showing papers in "Methods in Enzymology in 1982"
TL;DR: This chapter presents several techniques for further purification of the actin, finding that extensive dialysis results in eventual dissociation of actin-myosin complexes and appearance of myosin in the final product.
Abstract: Publisher Summary This chapter explores some of the pitfalls associated with actin purification and to clarify in some detail the correct use and expected result from each step of the widely used muscle actin purification procedure. Typical preparations use about 10 g of acetone powder. The steps include in the purification are following: extraction of acetone powder; filtration using sterile cheesecloth; centrifugation; polymerization; high salt wash: tropomyosin removal; sedimentation of filamentous actin; depolymerization. It is found that extensive dialysis results in eventual dissociation of actin-myosin complexes and appearance of myosin in the final product. Prolonged dialysis can also lead to observable actin proteolysis and should therefore be avoided. Final steps are Clarification of G-actinadn Polymerization. Depending upon individual technique, buffer purity, dialysis times employed, and so forth, the actin preparation can contain small amounts of contaminating protein, including proteolysed actin. Thus, the chapter presents several techniques for further purification of the actin.
1,207 citations
TL;DR: This chapter presents a procedure for the preparation of myosin and its sub-fragments from rabbit skeletal muscle, the most widely used protein in studies of muscle contraction.
Abstract: Publisher Summary This chapter presents a procedure for the preparation of myosin and its sub-fragments from rabbit skeletal muscle. The discussion is limited to rabbit skeletal muscle myosin because this particular myosin is the most widely used protein in studies of muscle contraction. At a salt concentration approximating physiological conditions, myosin aggregates to form bipolar thick filaments with a central bare zone; S1 heads protrude from the filament on either side of the bare zone. The insolubility of myosin at μ
875 citations
TL;DR: This chapter presents methods generally applicable to the preparation of native collagen from a variety of sources, conducted at relatively low temperatures, in the range of 4-8 °C, to minimizes bacterial growth, enhances the solubility of native collagens, and ensures the retention of native conformation on the part of the Solubilized Collagens.
Abstract: Publisher Summary This chapter presents methods generally applicable to the preparation of native collagen from a variety of sources. The preparation of a collagen sample generally involves several different steps. These include acquisition and preliminary processing of an appropriate tissue or organ, extraction of the collagen, and its purification. The latter process requires not only elimination of the noncollagenous components that are present in the extract, but may also require selective removal of alternative collagen types. This can usually be accomplished by a judicious use of selective precipitation techniques accompanied ultimately by one or two chromatographic steps. Given the extreme diversity of the tissues or organs in which collagen occurs as well as the multiplicity of collagen types that may be present in a given source, there understandably exists no single standard or preferred method for the preparation of collagen. Unless otherwise indicated, all procedures are conducted at relatively low temperatures, in the range of 4-8 °. This minimizes bacterial growth, enhances the solubility of native collagens, and ensures the retention of native conformation on the part of the solubilized collagens.
764 citations
TL;DR: This chapter discusses structure and procedure for isolation, and analysis of gangliosides, along with a gas-liquid chromatographic method that has the attributes, of specificity and sensitivity.
Abstract: Publisher Summary This chapter discusses structure and procedure for isolation, and analysis of gangliosides. Most widely used procedure for isolation is based on the ability of gangliosides to partition from the chloroform-methanol phase in which they are extracted into the upper water-enriched phase, leaving behind the bulk of other lipids. There is also a procedure based on DEAE-Sephadex. This method employs an anion-exchange resin to separate gangliosides first, along with other acidic lipids, from the much larger quantity of neutral and zwitterionic lipids that do not bind to the resin. The subsequent steps of base treatment, dialysis, and silicic acid chromatography are designed to remove the other acidic lipids and any acidic low molecular weight contaminants that accompany the gangliosides. Other types of chromatography is used for resolution. Quantitative estimation of gangliosides is usually based on the sialic acid moiety. Various procedures are outlined, along with a gas-liquid chromatographic method that has the attributes, of specificity and sensitivity.
700 citations
TL;DR: This chapter outlines the procedures for calculating the practical pKa values that apply to the buffer components under the chosen experimental conditions and the ionic strength of reaction mixtures at various pH values and hence the amount of inert electrolyte that must be added to maintain the ionsic strength constant.
Abstract: Publisher Summary This chapter outlines the procedures for calculating (a) the practical pKa values that apply to the buffer components under the chosen experimental conditions and (b) the ionic strength of reaction mixtures at various pH values and hence the amount of inert electrolyte that must be added to maintain the ionic strength constant. It also draws attention to the composition of two- and three-buffer systems that can be used over a wide pH range and whose ionic strength is essentially independent of pH. Although the basic theory relating to ionic strength is well known to physical chemists, it is not as familiar to biochemists. It is for this reason that a somewhat detailed presentation of the theory is given. Emphasis is placed on the use of pH studies for investigating enzyme-catalyzed reactions, but the same principles apply in connection with binding and other thermodynamic studies.
637 citations
TL;DR: Fibronectin is a cell-surface and blood glycoprotein that apparently mediates adhesion of cells to the extracellular matrix that may contribute to their capacity for invasive and metastatic growth.
Abstract: Publisher Summary Fibronectin is a cell-surface and blood glycoprotein that apparently mediates adhesion of cells to the extracellular matrix. Malignant cells tend to lack cell surface fibronectin, and this may contribute to their capacity for invasive and metastatic growth. Early isolation methods for plasma fibronectin made use of its propensity to precipitate with fibrinogen and some other proteins when plasma is allowed to stand in the cold. A large fraction of the plasma cryoprecipitate is fibronectin, and it can be purified from such precipitate by using a combination of differential precipitation steps and ion exchange chromatography. Fibronectin from the surface of cultured fibroblasts can be isolated by extraction with low concentrations of urea. Antifibronectin columns can also be used to isolate fibronectin from both plasma and cell cultures. These methods have now largely been replaced by affinity chromatography on gelatin-Sepharose. The methods used in immunochemical quantitation of fibronectin include radioimmunoassay, enzyme immunoassay, and rocket immunoelectrophoresis. Of these methods, enzyme linked immunosorbent assay is probably the most useful one.
537 citations
TL;DR: This chapter discusses the determination of binding stoichiometry by continuous variation method known as the job plot method, which is based on the measurement of complex formation at various combinations of mole fractions of the reactants.
Abstract: Publisher Summary An important element in the study of protein-ligand or protein-protein interaction is the binding stoichiometry of the reactants. This chapter discusses the determination of binding stoichiometry by continuous variation method known as the job plot method. Determination of stoichiometry is based on the measurement of complex formation at various combinations of mole fractions of the reactants. The chapter primarily concerned with the use of enzymic activity as an indicator of complex formation. In this sense, changes in enzymic activity are the same as other measurable parameters such as fluorescence or absorbance changes. The advantage of measuring activity is the relative ease of carrying out the experiments. It is, however, applicable only to ligands (or regulatory proteins) that are not consumed during the enzymic reaction—namely, the effectors, cofactors, and nonconsumable substrates. For purposes of comparison, other approaches that can utilize enzymic activity as an indicator of enzyme-ligand complex formation, such as the use of modified Klotz equation, are also discussed.
452 citations
TL;DR: The hydrazinolysis reaction is used for the isolation of whole asparagine-linked sugar chains as oligosaccharides by N-acetylation, instead of cleaving all glucosamine linkages by deamination.
Abstract: Publisher Summary Several enzymic and chemical methods liberate asparagine-linked sugar chains as oligosaccharides. Among them, endo-β-N-acetylglucosaminidases have been used to elucidate the whole structures of highmannose type of sugar chains and all of the sugar chains of hen egg albumin, and to confirm the presence of hybrid-type asparagine-linked sugar chains. However, the substrate specificities of these enzymes have hampered their use for the study of complex-type asparaginelinked sugar chains, a most important series of this group. The method described in this chapter is the chemical procedure applicable to the study of asparagine-linked sugar chains without any limitations. The hydrazinolysis reaction is used for the isolation of whole asparagine-linked sugar chains as oligosaccharides by N-acetylation, instead of cleaving all glucosamine linkages by deamination. For determining an optimal condition to release oligosaccharides, the hydrazinolysis products of bovine IgG and of gltcoprotein-IV obtained from hen egg albumin are carefully examined.
422 citations
TL;DR: The following fractions can be obtained after extraction of biological samples with ODS silica, if nonpolar PGs such as 13, 14-dihydro-15-oxo-PGE 2 are to be extracted, petroleum ether should be used.
Abstract: The following fractions can be obtained after extraction of biological samples with ODS silica. 1. 1. Arachidonic acid or other nonpolar fatty acids. Apply sample to ODS silica in acidified 30% ethanol. Wash with 30% ethanol (20 ml), water (20 ml), and petroleum ether (10 ml). Elute 20:4 with petroleum ether-chloroform (1:1, 20 ml). 2. 2. Monohydroxyeicosenoic acids. Apply sample in acidified 15–25% ethanol, depending on whether it is desired to obtain a subsequent fraction containing PGs. Wash ODS silica with the same concentration of ethanol (20 ml), water (20 ml), and petroleum ether (20 ml). Elute monohydroxyeicosenoic acids with petroleum ether-chloroform (65:35, 20 ml), chloroform (10 ml) or methyl formate (10 ml), depending on whether or not it is necessary to obtain a subsequent PG fraction. 3. 3. Prostaglandins and thromboxanes. Apply sample to ODS silica in acidified 15% aqueous ethanol. Wash with 15% ethanol (20 ml) followed by petroleum ether (20 ml) or, if it is desired to extract monohydroxyeicosenoic acids, water (20 ml), and petroleum ether-chloroform (65:35, 20 ml). If nonpolar PGs such as 13, 14-dihydro-15-oxo-PGE 2 are to be extracted, petroleum ether should be used. Elute PGs and Txs with methyl formate (10 ml). 4. 4. Monohydroxyeicosenoic acids, PGs, and Txs. Apply sample in acidified 15% aqueous ethanol. Wash ODS silica with 15% aqueous ethanol (20 ml), water (20 ml) and petroleum ether (20 ml). Elute with methyl formate (10 ml). 5. 5. Polar PG metabolites. Apply sample in acidified 0–10% aqueous ethanol (for ω-hydroxy-PGs 10% ethanol can be used). Wash ODS silica with the same concentration of ethanol (20 ml) followed by petroleum ether-chloroform (65:35). Elute PG metabolites with methyl formate (10 ml). 6. 6. PGs extracted from neutral or basic media. Apply sample to the ODS silica in 10–15% aqueous ethanol at the desired pH. Wash with the same concentration of ethanol (10 ml) (for greater speed, it should be possible to eliminate this step), followed by petroleum ether (20 ml). Elute with methyl formate (10 ml). For nonpolar products, better results might be obtained by washing the ODS silica with water prior to petroleum ether.
417 citations
TL;DR: Two methods for preparing tubulin are presented: purification by cycles of assembly and disassembly followed by chromatography on phospbocellulose and modified Weisenberg procedure, each of which yields several hundred milligrams of purified protein.
Abstract: Publisher Summary This chapter presents procedure for preparation of tubulin from brain. Two methods for preparing tubulin is presented (a) purification by cycles of assembly and disassembly followed by chromatography on phospbocellulose (b) purification by the modified Weisenberg procedure, each of which yields several hundred milligrams of purified protein. The principal properties of the tubulin prepared by the two methods appear to be identical and a choice of one route or the other can be made on the basis of available apparatus or of the investigator's familiarity with the manipulations involved. The tubulin that results from either preparation is substantially free of microtubule-associated proteins. The protein concentration of the solution is determined spectrophotometrically in 6 M guanidine hydrochloride at 275 nm with an absorptivity value of 1.03 ml/(mg cm). It is then adjusted to 50-60 mg/ml and rapidly frozen in small aliquots to be stored in liquid nitrogen. Under these conditions the protein can be stored for at least 14 days without observable loss of colchicine binding activity or of its ability to undergo in vitro microtubule assembly.
381 citations
TL;DR: This chapter describes the assay, purification, and properties of the enzymes catalyzing the unique intracellular modifications—i.e., the hydroxylases and hydroxylysyl glycosyltransferases.
Abstract: Publisher Summary This chapter describes the assay, purification, and properties of the enzymes catalyzing the unique intracellular modifications—ie, the hydroxylases and hydroxylysyl glycosyltransferases The main types of assays methods for hydroxylases
TL;DR: This chapter provides the physicochemical background for the use of solvent isotope effects in biochemical studies, with fairly complete derivations of the requisite algebraic expressions, an account of underlying assumptions, and a review of pertinent experimental and theoretical information.
Abstract: Publisher Summary This chapter discusses the solvent isotope effects on enzyme systems. The introduction of deuterium in place of protium in the hydrogenic sites of water, and its consequent exchange into some positions of enzymes and substrates produces solvent isotope effects on the kinetic and equilibrium constants associated with the enzymic reaction. These effects, usually expressed as ratios of the appropriate constants in the two isotopic solvents HOH and DOD, are useful in the study of reaction mechanism. The chapter provides (a) the physicochemical background for the use of solvent isotope effects in biochemical studies, with fairly complete derivations of the requisite algebraic expressions, an account of underlying assumptions, and a review of pertinent experimental and theoretical information. (b) to outline workable procedures for carrying out experiments in this area; and (c) to present the apparatus for interpretation of the results.
TL;DR: Gel permeation chromatography can be used more effectively than was previously believed because of the occurrence of structural rules in oligosaccharides of natural origin and the evidence that N-acetylhexosamine, hexose, and methylpentose residues behave differently because of size increases the usefulness of the method.
Abstract: Publisher Summary Development of various techniques to liberate sugar chains from glycoproteins, there is a need to fractionate oligosaccharides composed of 8-20 monosaccharides is increasing. Gel permeation chromatography is useful tool for this purpose, which separates oligosaccharides by their sizes and is limited in fractionating oligosaccharides as compared to paper chromatography. However, because of the occurrence of structural rules in oligosaccharides of natural origin, the method can be used more effectively than was previously believed. The evidence that N-acetylhexosamine, hexose, and methylpentose residues behave differently because of size also increases the usefulness of the method. Moreover, careful analysis of the mobilities of various oligosaccharides has disclosed that even isomeric oligosaccharides show slight differences in their mobilities on the column, and these differences can be correlated with the mobilities of particular disaccharide structures within each oligosaccharide.
TL;DR: This chapter presents procedure for preparation of troponin and its subnits for both skeletal and cardiac muscle, and there are significant differences between the purification procedures for the cardiac and skeletal troponins.
Abstract: Publisher Summary This chapter presents procedure for preparation of troponin and its subnits. The procedures presented are for both skeletal and cardiac muscle. Although there are significant differences between the purification procedures for the cardiac and skeletal troponins, the same basic procedures are employed. The first step consists of making an ether powder (procedure is identical for both cardiac and skeletal muscle), followed by an high ionic strength extraction and initial fractionation of troponin. This partially purified troponin, called crude troponin, can be further purified to yield pure troponin using Cibacron Blue-Sephacryl chromatography similar to the Cibacron Blue-agarose chromatography procedure. The crude troponin can also be used to prepare pure troponin subunits by various combinations of chromatography on DEAE-Sephadex and CM-Sephadex in the presence of 6 M urea and 1 mM EDTA.
TL;DR: This chapter discusses the preparation of retinal rod outer segments and the assay of their purity and several factors to be carefully considered, including the shearing forces employed during initial and subsequent homogenization.
Abstract: Publisher Summary This chapter discusses the preparation of retinal rod outer segments. Isolation of rod outer segments (ROS) and the assay of their purity must reflect the research goals that require their isolation. It is not sufficient to apply one procedure in rote fashion that was initially designed for one purpose and use the circular argument that the presence or absence of some component in the isolated membrane fraction is evidence for or against the existence of that component in the organelle in vivo . There are several factors to be carefully considered while modifiing each step: (1) the shearing forces employed during initial and subsequent homogenization; (2) the ionic strength, ionic composition, and osomolality of each of the media; (3) the presence or absence of reducing agents; (4) the presence or absence of nucleotide phosphates; (5) the possible addition of proteolytic inhibitors; and (6) the degree of light exposure of the retina and of isolated ROS.
TL;DR: The information concerning the distribution and localization of the collagens in various connective tissues strongly support the notion that each of thecollagens has evolved to accommodate specific physiological requirements of organisms of increasing complexity.
Abstract: Publisher Summary This chapter outlines the development of information concerning the molecular heterogeneity of collagen. This heterogeneity is ultimately determined by the genome, but is amplified by posttranslational events that allow for wide variations in the extent to which different types of procollagen molecules are processed prior to incorporation into their respective fibrous elements. The extent to which procollagen molecules are processed more than likely specifies the general properties of the fibrous elements formed at any given site. The general properties of the fibrous elements may, in turn, be of great importance with respect to specific interactions with other matrix macromolecules as well as with cells. The information concerning the distribution and localization of the collagens in various connective tissues strongly support the notion that each of the collagens has evolved to accommodate specific physiological requirements of organisms of increasing complexity. In this regard, it is likely that the multiple genes for collagen synthesis represent an informational multigene family, or perhaps a number of families, the existence of which allows the selective synthesis of discrete, albeit functionally related, proteins.
TL;DR: This chapter presents the procedure for preparation and identification of α- and β-Tropomyosins, and it is shown that the nucleic acid can be largely removed by ion-exchange chromatography on DEAE-cellulose at pH 7.6, and this procedure is recommended as a final step in the purification of the mixed chains of tropomyosin if nucleic acids contamination is indicated.
Abstract: Publisher Summary This chapter presents the procedure for preparation and identification of α- and β-Tropomyosins The preparation involves the initial removal of the soluble sarcoplasmic proteins by extraction of a muscle mince with water or dilute salt solutions and the dehydration of the insoluble residue by washes with alcohol and ether Acetone is often now substituted for ether Myosin is denatured by these procedures The tropomyosin is then extracted from the dried ether or acetone powder with 1 M KC1 at neutral pH and purified by repeated cycles of isoelectric precipitation at pH 45-47 and ammonium sulfate fractionation at pH 70-78 At this stage the product is sometimes contaminated with low levels of troponin components or their proteolytic degradation products These may be conveniently removed by chromatography on hydroxyapatite in 1 M KC1 with a phosphate gradient at pH 70 When the methods described are used with muscles from various sources, the recovered tropomyosin appears to be sometimes con taminated with significant amounts of nucleic acid It is shown that the nucleic acid can be largely removed by ion-exchange chromatography on DEAE-cellulose at pH 76, and this procedure is recommended as a final step in the purification of the mixed chains of tropomyosin if nucleic acid contamination is indicated
TL;DR: Application of the various techniques described above has identified the different classes of proteoglycans in a starting extract based upon differences in charge density, hydrodynamic size, and buoyant density.
Abstract: Publisher Summary This chapter presents procedure for extraction and characterization of proteoglycans In general, tissue or cultures should be processed for extraction quickly and using procedures that minimize endogenous degradation Fresh tissue should be chilled on ice, cleaned, and minced quickly Extraction efficiency is often improved when finer tissue pieces are used to expose more surface area to the solvent Once the proteoglycans are in solution, different fractionation procedures (ion exchange in 7 M urea, rate zonal-velocity, centrifugation molecular-sieve chromatography, electrophoresis in composite polyacrylamide-agarose gels), which take advantage of their macromolecular properties, are used to purify them from other macromolecules in the extract and to begin to separate them from each other Application of the various techniques described above has identified the different classes of proteoglycans in a starting extract based upon differences in charge density, hydrodynamic size, and buoyant density Such analyses not only provide information about the physical properties of the proteoglycans but help design effective strategies for purifying at least some of the subpopulations of proteoglycans identified
TL;DR: This chapter presents procedure for purification and characterization of PAI and PAII, which involves crude extraction from bacteria grown in Grelet's medium, ammonium sulfate precipitation, and chromatography on Sepharose 4B.
Abstract: Publisher Summary Extracts of certain strains of Pseudomonas aeruginosa contain two lectins: PAI and PAII. This chapter presents procedure for purification and characterization of PAI and PAII. The PAI purification involves crude extraction from bacteria grown in Grelet's medium, ammonium sulfate precipitation, and chromatography on Sepharose 4B. The yield of the purified PA-I is relatively high and is practically free of PA-II. PA-II is purified from the extracts of the bacteria grown in nutrient broth. The first two steps of its purification are the same as for PA-I: heating to 70 ° and precipitation by ammonium sulfate, as described above. The dissolved ammonium sulfate precipitate is purified by affinity chromatography on D-mannose-bearing Sepharose 4B. Thirty milliliters of the lectin preparation are loaded onto a column (4 x 20 cm) containing the modified Sepharose. The activity of the lectins is determined by measuring their ability to agglutinate neuraminidase or papain-treated human erythrocytes. The hemagglutination assay can also be used as an indirect demonstration of lectin interaction with nonagglutinable cells by determining the decrease in the hemagglutinating activity of the lectin preparations as a result of their exposure to the examined cells. The interaction of PA-II with cells can also be detected by a peroxidase-binding assay.
TL;DR: A further result is that the fate of oxygen in the catalysis can be followed and the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate is revealed.
Abstract: Publisher Summary This chapter reviews the application of an 18 O exchange method to study the catalysis of the hydration of CO 2 by carbonic anhydrase, which, with a maximal turnover number near 10 6 sec –1 , is one of the fastest known enzymic reactions. The catalysis is so rapid that proton transfer steps between the enzyme and its environment, which are not ordinarily rate limiting for slower enzymic reactions, become rate-limiting and more amenable to investigation with carbonic anhydrase. The 18 O exchange method is chosen to investigate this problem because it is an equilibrium method that may be used in the absence of buffer or in the presence of excess buffer concentration, and buffers are sources of protons for transfer to the enzyme. With this method, it is possible to elucidate the role of proton transfer in the pathway of the catalytic hydration of CO 2 . A further result is that the fate of oxygen in the catalysis can be followed; hence, this method reveals the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate.
TL;DR: This chapter is an introduction to the properties and prospective applications of the stable complex formed by the binding of ADP and vanadate ion to the myosin active site, as well as a discussion of techniques for its synthesis and analysis.
Abstract: Publisher Summary It is possible to obtain stable complexes of myosin that mimic transient intermediates of ATP hydrolysis. One such model complex, which resembles the central myosin-products intermediate (M • Pr), is formed by the binding of ADP and vanadate ion to the myosin active site. This chapter is an introduction to the properties and prospective applications of the complex, as well as a discussion of techniques for its synthesis and analysis. The stable complex is best understood in the context of the properties of vanadate ion (i.e., orthovanadate), which is a tetrahedral oxyanion of pentavalent vanadium. By virtue of its geometry, size, and charge, vanadate (abbreviated Vi) is an analog of phosphate ion. In preparation of vanadate-trapped myosin either subfragment-1 or heavy meromyosin is generally used for chromatic separation, although whole myosin readily undergoes modification. Vanadate alone forms a weak reversible complex with myosin, much like phosphate. Yet when ADP is included, a stable, enzymically inactive complex is formed (M† • ADP • Vi). A typical preparation of M† • ADP • Vi is carried out with a myosin site concentration of 20 μM in 0.09 M NaCl, 5 mM MgCl2, and 20 mM Tris, pH 8.5. Ionic strength is kept low and pH high to facilitate separation of M† • ADP • Vi by ion-exchange chromatography. Preparation can be carried out at pH 7 with equal ease, although subsequent separation is more complex.
TL;DR: A rapid way to analyze radioactively labeled 3- and 4-hydroxyproline in a sample without resorting to an amino acid analyzer is the method of separation using high-voltage electrophoresis on paper.
Abstract: Publisher Summary This chapter describes assays for determining unlabeled or labeled trans-4-hydroxyproline and cis-4-hydroxyproline as well as two assays that can distinguish trans-3- from trans-4-hydroxyproline. 4-hydroxy-L-proline can be measured by colorimetric reaction that involves oxidation of hydroxyproline to pyrrole. This assay for hydroxyproline is applicable to the detection of hydroxyproline in a variety of connective tissues, cells in tissue culture, and purified collagens. Moreover, the radiochemical assay is based on the oxidation of hydroxyproline to pyrrole except that the hydroxyproline is converted to radioactive pyrrole, which can then be quantified using scintillation counting. An internal standard of unlabeled hydroxyproline is added to each sample prior to analysis, and its recovery is determined to correct the values obtained for the hydroxy[ 14 C]proline. In addition, a rapid way to analyze radioactively labeled 3- and 4-hydroxyproline in a sample without resorting to an amino acid analyzer is the method of separation using high-voltage electrophoresis on paper.
TL;DR: This chapter describes the methods that give the best results in terms of coating plain plastic plates and making collagen gels as substrates for cell culture and presents evidence on the effects of collagen, using examples from normal hepatocytes and hepatoma cells.
Abstract: Publisher Summary This chapter describes the methods that give the best results in terms of coating plain plastic plates and making collagen gels as substrates for cell culture and presents evidence on the effects of collagen, using examples from normal hepatocytes and hepatoma cells. Regardless of the particular type of collagen gel technique, all collagen gel preparations can be processed by the usual histological techniques for embedding and sectioning. The collagen gels can be fixed in formaldehyde or in ethyl alcohol. Alternatively, the cells can be removed from the gel by treatment with collagenase and processed separately. Subtle changes can be seen when one compares cells grown on collagen-coated plates with cells grown on floating collagen gels. With hepatocytes, it is found that cells growing on floating collagen gels remained viable longer and also maintained inducibility of characteristic hepatocellular enzymes for longer times in culture. When hepatocytes are grown on collagen-coated plates they rapidly lose the levels of cytochrome P-450 within 72 hr. To investigate possible reasons for the differences in behavior of the cells on different types of collagen substrates, the chapter compared hepatocytes grown on collagen-coated plates and collagen gels with hepatocytes grown on confluent monolayers of human fibroblasts.
TL;DR: Many chromatographic procedures can be employed for analysis of prostaglandins, but thin layer chromatography has much to commend it in terms of efficiency, simplicity, and economy of money and effort.
Abstract: Publisher Summary This chapter discusses the extraction and thin-layer chromatography of arachidonic acid metabolites. In some cases prostaglandins may be determined directly in aqueous samples (e.g., radioimmunoassay or bioassay of tissue perfusates and cell culture fluids) without recourse to extraction, but more usually, extraction into an organic solvent is a prerequisite to separation and/or quantitation. The advantages of extraction are that it eliminates protein, imparts some specificity to the assay and, by concentrating material, improves the sensitivity of the analysis. The choice of any particular extraction technique is governed by its efficiency, specificity, reproducibility, and practicability. The extraction procedures efficiently remove all fatty acids, prostaglandins, and other hydroxy acids, but obviously if specific analysis of a particular compound is required some further purification is necessary. Many chromatographic procedures can be employed for analysis of prostaglandins, but thin layer chromatography has much to commend it in terms of efficiency, simplicity, and economy of money and effort.
TL;DR: This chapter describes several assays of the polymerization of actin, which vary widely in terms of their cost, ease of performance, time, sensitivity, and information provided.
Abstract: Publisher Summary This chapter describes several assays of the polymerization of actin. The assays vary widely in terms of their cost, ease of performance, time, sensitivity, and information provided. In some assays the actin solution is undisturbed, and in others the sample is sheared. Some assays use native actin, and others employ actin that has been covalently modified with a label. In capillary viscometry assay, viscosity is measured by the time required for a solution to pass through a glass capillary tube. Other methods to measure viscosity include rotational devices. It is reported that the ultraviolet absorption spectrum of actin filaments is different from that of actin monomers. The difference spectrum was positive from 240 nm to 200 nm, with a relative maximum at about 230 nm. This principle is used for the for analysis of actin. In addition, polymerization of actin can be assayed using flow birefringence which is based on the orientation of actin filaments in fluid flow. The sample is placed between two concentric cylinders, one of which rotates. Laminar flow in the sample tends to align the filaments in the direction of flow. This alignment is detected by refraction of polarized light. Finally, the chapter discusses how to measure various parameters associated with actin polymerization. We have separated into an accompanying chapter assays of the interactions among actin filaments.
TL;DR: It is found that the comprehensive immunochemical characterization of various antibodies against collagens and procollagens has provided the basis for their successful use in numerous other studies.
Abstract: Publisher Summary During the past decade antibodies have become important tools for characterizing structural, biological, and pathological properties of collagens. The most striking achievements are the development of reagents capable of distinguishing among various types of the protein as well as precursor-specific portions of procollagens. It was also possible to identify the structure of individual antigenic determinants, thus allowing the use of such reagents at the submolecular level. This chapter discusses methodological and applied aspects. It is found that the comprehensive immunochemical characterization of various antibodies against collagens and procollagens has provided the basis for their successful use in numerous other studies. Major applications of such reagents included the visualization of various types of collagens in tissue sections and cultured cells, the identification of metabolically labeled products by immunoprecipitation, and the development of sensitive quantitative assays for experimental and clinical studies.
TL;DR: In this chapter, it is assumed that a mathematical model (rate equation) should be fitted by nonlinear regression analysis to a set of experimental data and most procedures use the principle of least squares.
Abstract: Publisher Summary The fitting of rate equations to kinetic data in enzymology is an application of the treatment of experimental data in general and the use of mathematical models for quantitative description. By using statistical methods, a certain degree of objectivity is ascertained insofar as all investigators should get the same analytical results once they have agreed on the techniques to use. Certain statistical fitting procedures also provide quantitative measures of goodness of fit and of the reliability of the kinetic constants estimated, facilitating evaluation of the results and testing of hypotheses. This is the case for nonlinear regression analysis based on the principle of least squares. In this chapter, it is assumed that a mathematical model (rate equation) should be fitted by nonlinear regression analysis to a set of experimental data. Most procedures use the principle of least squares. While discrimination between rival mathematical models, two questions arise: do the models adequately describe the data? Is one model better than the other is? The first question may be answered by evaluating the results of the regression by the criteria for goodness of fit. If both models are adequate and fit the data equally well, the simplest model is chosen. However, independent information obtained by additional kinetic studies or by completely different experimental methods should be included in the discrimination procedure.
TL;DR: This chapter discusses the purification of rhodopsin by concanavalin a affinity chromatography, a technique based on the reversible binding of a ligand to a protein that represents one of the major advances in protein purification methodology.
Abstract: Publisher Summary This chapter discusses the purification of rhodopsin by concanavalin a affinity chromatography. Affinity chromatography represents one of the major advances in protein purification methodology. This technique depends on the reversible binding of a ligand to a protein. Rhodopsin has been identified as a mannose-containing glycoprotein and as such has the potential ligand properties for affinity chromatographic purification. The procedure to be described employs the nonionic detergent octyl glucoside. Rhodopsin fractions are pooled and can be concentrated by ultrafiltration using a Diaflo PM 10 or PM 30 filter. Pooled samples are generally concentrated to about 3 mg/ml of rhodopsin, involving a reduction in volume from about 40 ml to 5 ml. This process results in a concentration of octyl glucoside and rhodopsin, the final detergent concentration being in the range of 60 mM to 70 mM; this raises no concern with respect to rhodopsin stability, as rhodopsin is stable in octyl glucoside solutions as high as 300 mM.
TL;DR: This chapter discusses radioimmunoassay of the major plasma metabolite of PGF 2α, 15-Keto-13,14-dihydro-PGF 2α and the polyethylene glycol method, which gave the most reliable results.
Abstract: Publisher Summary This chapter discusses radioimmunoassay of the major plasma metabolite of PGF2α, 15-Keto-13,14-dihydro-PGF2α. In this particular assay, plasma volumes of as much as 0.5 ml may be assayed without disturbing interferences from other plasma constituents. After the addition of the labeled ligand, the tubes are left for incubation. The next step is the separation of the free and the antibody-bound fractions. Of all methods tried, 3 the polyethylene glycol method gave the most reliable results. The tubes are vigorously vortexed, the formed grayish-white pellet contains the precipitated γ-globulins including the antibody-bound fraction of the prostaglandin metabolite, whereas the free fraction is found in the supernatant. The yellow color of unextracted plasma gives about 10% reduction in counting efficiency in the scintillation counter as compared to the standard vials without plasma. It is important that the counted values for counts per minute are corrected for quenching. This can easily be done if the scintillation counter is equipped with automatic quench correction facilities, such as automatic external standard channels ratio. Before setting up routine radioimmunoassay runs, the optimal titer of the antibody must be established. This should always be done also with the commercial preparations, as experience shows that the recommended procedure from the manufacturer is often far from optimal.
TL;DR: This chapter presents a procedure for the extraction of lipids of purple membrane from extreme halophiles and of methanogenic bacteria.
Abstract: Publisher Summary This chapter presents a procedure for the extraction of lipids of purple membrane from extreme halophiles and of methanogenic bacteria. For lipid extraction, cells of Halobacterium cutirubrum or Halobacterium halobiurn are first grown aerobically at 37° and whole cells are extracted. A suspension of purple membrane (PM) is diluted with methanol and stirred in the dark. The mixture is centrifuged and the supernatant is collected. After centrifugation the combined supernatants are diluted with CHCl 3 and KCI with gentle mixing and the phases are allowed to separate in a separating funnel for several hours. The lower chloroform phase is removed, diluted with benzene and ethanol, and brought to dryness on a rotary evaporator. The residual total lipids are dissolved in chloroform to a known volume and subjected to separation and analysis. To extract lipids of Methanospirillurn hungatei, a suspension of cells is diluted with methanol and chloroform and the mixture stirred. The mixture is then centrifuged and the supernatant is collected. The pellet is suspended in NaC1 and extracted then supernatants are diluted with chloroform and distilled water in a 4-liter separatory funnel, mixed gently, and left at room temperature for several hours to allow the phases to separate. The chloroform phase is then removed, diluted with benzene and 99% ethanol, and brought to dryness on a rotary evaporator at 30–35 °. The residual lipids are dissolved in chloroform and stored at –20 °.