Showing papers in "Methods in Enzymology in 1988"

Lignin peroxidase of Phanerochaete chrysosporium

Abstract: Publisher Summary Several procedures have been described for growing Phanerochaete chrysosporiurn for ligninase production. These procedures differ somewhat in the medium formulation and types of growth vessels: (1) shallow stationary cultures, (2) agitated liquid cultures, and (3) rotating biological contactors (RBCs; disk fermenters). The recently developed use of agitated culture for production of ligninase permits easier “scale up.” Although ligninase can be produced in agitated flask cultures, the reliable use of stirred tank fermenters awaits further development, which is ongoing in several laboratories. This chapter describes the production of ligninase in shallow stationary cultures and in agitated cultures. Shallow stationary cultures (10 ml) are grown in rubber-stoppered, 125-ml Edenmyer flasks at 39 ° under 100% oxygen. The stationary cultures give somewhat more reliable and reproducible results than the agitated cultures.

Methods for measuring cellulase activities

Abstract: Publisher Summary The physical heterogeneity of the substrate and the complexity of the cellulase enzyme system present those wishing to measure cellulase activities with formidable problems Faced with the use of substrates that are ill defined, and an enzyme system that consists often of a multitude of enzymes acting in synergism in a manner not yet fully understood, the enzymologist has developed a bewildering number of assays in an attempt to throw some light on the complex enzymatic interactions involved in the breakdown of cellulose This state of affairs, compounded by the existence of a plethora of arbitrary units of activity, has made comparison of quantitative data obtained in various laboratories impossible This chapter describes a number of alternative assay methods that may help toward a better evaluation of cellulase components and their complex interactions that result in the solubilization of cellulose Some of these alternative methods are included for practical considerations and many are included because of the nature of the unresolved problems

Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands.

Abstract: Publisher Summary This chapter focuses on the computer programs for calculating total from specified free, or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Many experiments in biology, biochemistry, biophysics, and physiology require aqueous solutions with concentrations of free cations, far below those obtainable by simply adding the corresponding salts to the solutions. Then metal buffers made up of the metallic ion (or cation) and the appropriate ligand (or anion) must be used. Similarly, living cells contain ligands, such as cation-binding proteins, that cause the total to differ from the free concentrations of metals. Ligands also bind hydrogen ions so that instead of a single complex between metal and ligand, several complexes form, corresponding to various degrees of protonation. Each has its own stability constant. Thus, apparent stability constants (K app ) have to be calculated to characterize the multiple equilibria among one metal, one ligand, and the hydrogen ion at a given pH value. The main thrust of the present chapter is to describe a FORTRAN program named "SPECS" in such a way that it can be readily used by the largest number of investigators possible.

[85] Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803

Abstract: Publisher Summary A new genetic technique has been developed in Synechocystis 6803 for the molecular analysis of electron transport in the photosystem II reaction center (PSII). This methodology permits specific PSII genes to be deleted from the cyanobacterial genome. The deleted genes can then be replaced with copies modified by site-directed mutagenesis. In this way, specific amino acid changes can be engineered in the polypeptides that bind the pigments and electron carriers in the PSII protein complex. No comparable methodology for the molecular analysis of PSII is available in higher plants. This experimental system depends on two important characteristics of Synechocystis 6803 found in few other cyanobacteria: a naturally occurring genetic transformation system and the ability to grow photo-heterotrophically on glucose, which is necessary for the propagation of PSII mutants that are incapable of photosynthesis. PSII is functionally and structurally similar in the chloroplasts of higher plants and in cyanobacteria. The chapter describes a procedure for deleting PSII genes from Synechocystis 6803 to create a PSII mutant; replacement of the deleted genes to restore photosynthetic function; and some of the properties of the genetic transformation system in this cyanobacterium.

Isolation and purification of cyanobacteria.

Abstract: Publisher Summary This chapter focuses on the isolation and purification of Cyanobacteria. Cyanobacterial populations recognized in their natural habitat should be sampled with sterile instruments and placed in sterile containers to ensure the origin of eventual isolates. If funds permit, commercial sterile disposable scalpels, pipets, and plastic tubes are very convenient for this purpose. Only small quantities (a pea-size equivalent generally being ample) are required from habitats where macroscopic growth is visible. Sampling of endosymbiotic cyanobacteria from coralloid nodules of Cycadaceae or the stems of Gunnera can be performed as described for soil and rock-borne cyanobacteria, but other host-cyanobacteria associations might require more special treatments. To isolate cyanobacteria from lakes and ponds in which cyanobacterial growth is not visible with the eye (or even after examination with a portable microscope) it is advisable to take larger samples: 250- to 500-ml sterile screw-cap centrifuge pots, filled almost completely with sampling water, are convenient containers for transport and allow immediate concentration (by centrifugation) on arrival in the laboratory, the sampling volume generally being sufficient to isolate cyanobacteria present even in only low numbers. The origin of many cyanobacteria currently in culture is poorly characterized because little more is known about their habitat than that they were derived from a soil sample, freshwater, or marine environment, which is rather restricted information (although better than source unknown, another not uncommon description).

Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples.

Abstract: Publisher Summary This chapter discusses the rapid colorimetric micro-method for the quantitation of complexed iron in biological samples. The array of methods available for quantitating iron in an experimental sample often bewilders the investigator occasionally faced with the need for such quantitation. However, because of the development of more sensitive chromophoric chelators and simpler techniques, new methodologies continually evolve. This chapter describes one of these more recent methodologies for the quantitative determination of iron with ease, sensitivity, and simplicity. Methods commonly employed for the quantitation of complexed iron in biological samples rely on an initial treatment, which releases the complexed iron for its subsequent quantitative determination. Dry ashing in a furnace or wet ashing with hot concentrated acid releases the iron, but both procedures take time and present hazards, particularly for the researcher with only an occasional need to determine the iron content of a biological sample. This chapter explains that the simplicity of the assay procedure easily accommodates a preliminary investigation to examine for possible interference or contamination by any buffer system under consideration. In conclusion, the procedure detailed in this chapter offers the investigator without access to more sophisticated instrumentation, a simple and sensitive colorimetric method for the quantitation of iron in biological material.

Conjugal transfer of DNA to cyanobacteria.

Abstract: Publisher Summary This chapter focuses on the conjugal transfer of DNA to Cyanobacteria. Conjugation appears to be a general means to introduce DNA from Escherichia coli into cyanobacteria, using the broad host range conjugal apparatus of an IncP plasmid, such as RP4. RP4, originally isolated from Pseudomonas, has been shown to mediate the transfer of DNA into a wide range of gram-negative bacteria, including such distantly related organisms as myxobacteria, thiobacilli, and unicellular and filamentous cyanobacteria. To obtain stable conjugal transfer it is necessary that (1) conjugal contact be made, (2) transferred DNA escape restriction or degradation, and (3) the DNA replicate autonomously or integrate into one of the replicons of the recipient. The first requirement is probably met in the great majority of gram-negative eubacteria, cyanobacteria included. The task for the experimenter is to find conditions that permit the last two requirements to be met as well. Even with transformable unicellular cyanobacteria, conjugation may be the preferred route of DNA transfer in certain cases. DNA taken up by unicellular cyanobacteria appears to be randomly cut early during the transformation process, so that the efficiency of transfer of a segment of nonhomologous DNA decreases exponentially with its length.

Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension

Abstract: Publisher Summary This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Chemical and enzymatic probing, monitored by primer extension, has become a powerful tool for the analysis of RNA structure. The reactivities of individual nucleotides composing large RNA molecules may be determined rapidly by utilizing a series of primers spaced at approximately 200 nucleotide intervals. In addition, numerous chemical reagents and nucleases may be employed as probes, since the only requirement is that they modify the template so as to produce pauses or stops in the progress of reverse transcriptase. The RNA, either alone or complexed with proteins and/or ligands, is incubated under suitable conditions with chemical or enzymatic probes. Dimethyl sulfate (DMS), kethoxal (KE) and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatic probes. The extent of the reactions is limited so that no more than a few stops are present within 300 nucleotide stretches in a given RNA molecule.

Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle.

Abstract: Publisher Summary This chapter describes the purification of two forms of protein phosphatase- 1 (PP-I 1 and PP-l G ), and three forms of protein phosphatase-2A (PP-2A 0 , PP-2A 1 , and PP-2A 2 ) from skeletal muscle. Procedures for isolating the free catalytic subunits (termed PP-l c and PP-2A c ) are documented and the structures of these enzymes are summarized in the chapter. The free catalytic subunits are isolated by a modification of the procedure of Lee and co-workers. Skeletal muscle extracts prepared from 3000 g of muscle are adjusted to pH 7.2 with 10 M ammonium hydroxide, and 350 g of solid ammonium sulfate are added to each liter of solution to bring the degree of saturation to 55%. After standing for 30 min the suspension is centrifuged for 40 min at 4200 g and the supernatant discarded. The PP-2A c from poly(L-lysine)-Sepharose is purified in an identical manner to PP-I c .

[27] Extension inhibition analysis of translation initiation complexes☆

Abstract: Publisher Summary This chapter presents extension inhibition to the study of translation initiation complexes. The chapter also describes the method of using bacteriophage T4 gene 32 mRNA. Early experiments showed that ribosomes, in the presence of fMet-tRNA Met f could properly select translation initiation regions of bacteriophage RNA and protect these regions from ribonuclease digestion. This chapter subsequently demonstrated that a 70S–fMet-tRNA Met f complex, bound to the coat protein initiation site on Q β plus strand RNA, cannot be dislodged by Q β replicase engaged in negative strand synthesis. It was reasoned that specific binding of ribosomes (30S or 70S) to mRNA might lead to pausing or termination of reverse transcriptase when a bound ribosome is encountered. In extension inhibition, a 5'- 32 P-end-labeled oligo deoxyribonucleotide, complementary to a region on the mRNA that is 3' to the initiation codon, is annealed to either total cellular RNA or to a purified transcript. This [ 32 P]oligonucleotide-mRNA hybrid is then incubated with ribosomes, and the complexes analyzed by primer extension.

[3] Culturing methods for cyanobacteria

Abstract: Publisher Summary This chapter focuses on the culturing methods for cyanobacteria. For the establishment of new cultures, as much ecological information as possible about the field populations should be gathered and recorded. With this, it eventually becomes possible to relate diverse information obtained from culture strains to the survival strategies of the organism in nature, ultimately the object of all biological research unless it is directed solely to the benefit of humans. Cyanobacteria, unlike most other prokaryotes (eubacteria or archaebacteria) are usually seen and often recognizable (even at the species level) before collection; they are present in many cases as plankton blooms or as dense tufts or mats which are composed of few other species. Only a miniscule percentage of cyanobacterial species have been brought into culture. This, in part, reflects the relatively few and mainly recent attempts to isolate and grow diverse cyanobacteria. Cyanobacteria from certain habitats and those of certain taxonomic groups seem to be extremely recalcitrant with regard to being cultured with present methods. Identification of cyanobacteria, whether of natural populations or of cultured material, is not a simple task, because at present, there are three major systems of classification: Geitler, Drouet, and Stanier systems.

[34] Complementary chromatic adaptation: Physiological conditions and action spectra

Abstract: Publisher Summary This chapter focuses on the physiological conditions and action spectra of complementary chromatic adaptation. Photosynthetic organisms can modulate their relative pigment content in response to changes in either light intensity or light wavelength. Generally, one observes an inverse correlation between light intensity and pigment content—the less light energy available, the more photosynthetic pigments are synthesized by the cells. The effect of light wavelength on the pigment content of the cells, termed complementary chromatic adaptation, appears to be restricted to some cyanobacteria. In this type of adaptation, changes in cell pigmentation in response to specific spectral illuminations result from modifications of the relative amounts of the red-colored phycoerythrin (PE) and the blue-colored phycocyanin (PC), with a predominance of PE in green-light-grown cells and of PC in red-light-grown cells. The regulatory processes involved in complementary chromatic adaptation are controlled by a photoreceptor pigment system which presumably acts at the transcriptional level. The property of photoreversibility of the cyanobacterial photoreceptor resembles that of the phytochrome present in higher plants and algae, although its action maxima are situated at shorter wavelengths in the visible spectrum.

Preparation of crystalline, amorphous, and dyed cellulase substrates

Abstract: Publisher Summary The number of cellulose substrates used to determine cellulase activity is vast. This chapter discusses only those substrates that are either widely used or are of special value in some assay procedure. The chapter focuses on the preparation of crystalline, amorphous, and dyed cellulase substrates. Many cellulase substrates that are extensively used are commercially available. These include filter paper, Avicel, Sigmacel, Solka Floc, carboxymethylcellulose, and hydroxyethylcellulose.

Assay methods for pectic enzymes

Abstract: Publisher Summary The characterization and purification of a pectic enzyme is often complicated by the presence of other pectic enzymes produced by the source microorganism. This chapter describes three assay procedures that address this problem. Pectic enzymes from Erwinia spp. is used as examples in the chapter. The assays are readily adapted to the analysis of pectic enzyme complexes of other organisms and can be used with crude preparations. Results from plant tissue extracts should be interpreted cautiously, however, because of the prevalence of pectic enzyme inhibitors. The activity stains in the first procedure enable rapid approximation of the number and type of pectic enzymes in the sample and can guide purification strategies, particularly when used in conjunction with titration curves. The second and third procedures permit quantitative assay of hydrolytic and β-eliminative pectic enzymes, respectively. The action patterns of purified pectic enzymes are determined by viscometric and reaction product analyses; oligogalacturonides are resolved by paper chromatography or thin-layer chromatography and detected with bromphenol blue or thiobarbituric acid spray reagent.

The Na,K-pump

Abstract: The Na-K pump operates by a stepwise change in Na+ versus K+ affinities and a gating reaction governed by the reaction with ATP. The activity is regulated by the intra- as well as the extr...

Manganese peroxidase of Phanerochaete chrysosporium: Purification☆

Abstract: Publisher Summary This chapter discusses a method for purification of the manganese peroxidase of P. chrysosporium. It discusses assay methods for manganese. Manganese peroxidase may be assayed using a variety of aromatic substrates, particularly those that are employed for assays of common peroxidascs such as horseradish peroxidase. Reaction mixtures, however, must be supplemented with Mn(II) ions. A very convenient assay of manganese peroxidase activity involves monitoring the enzyme's oxidation of Mn(II) to Mn(III). This assay is best used with purified preparations of the peroxidase, as contaminating metals such as iron and copper inhibit the reaction. The most convenient assay for ligninase is a spectrophotometric assay that monitors the oxidation of veratryl alcohol to veratryl aldehyde.

Colorimetric assay for chitinase

Abstract: Publisher Summary This chapter describes the colorimetric assay for chitinase, which is applicable to the various types of chitinase present in microorganisms, animals, and plants The chapter evaluates it with particular reference to plant chitinases, because they may function as a defense against chitin-containing pathogens The most widely used colorimetric assay for plant chitinases has been an exochitinase assay, based on the determination of monomeric N-acetylglucosamine (GlcNAc) released from colloidal chitin However, plant chitinases generally are endochitinases and produce chitooligosaccharides as principal products Therefore, measurements of plant chitinases with the exochitinase assay should be viewed with caution For accurate determination, it is essential to measure the chitooligosaccharides produced in the assay This can be accomplished by the enzymatic hydrolysis of the reaction products to monomeric GlcNAc prior to the colorimetric measurement The chapter compares the assay with other chitinase assays

H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: purification and reconstitution.

Abstract: Publisher Summary This chapter focuses on the purification and reconstitution of H + -ATPase from plasma membranes of Saccharomyces cerevisiae and arena sativa roots The plasma membranes of fungi and plants contain a similar ATPase, which represents a novel type of proton pump Two convenient sources for these enzymes are bakers' yeast ( Saccharomyces cerevisiae ) and oat (Arena sativa) roots The purified plasma membrane ATPases from these organisms can be reconstituted into proteoliposomes that catalyze ATPdriven proton transport A summary of the purification of the yeast and oat root plasma membrane ATPases is shown in the chapter The evaluation of the purifications is complicated by the activating and inactivating effects of detergents Detergent activation results from unmasking latent enzyme molecules present in closed vesicles, while detergent inactivation may be caused by delipidation of the enzymes The purified plasma membrane ATPase from yeast and oat roots contain a major polypeptide of about 105 kDa, which represents more than 70–80% of the protein in the preparations Some minor polypeptides of lower molecular weight are usually present in variable and less than stoichiometric amounts

Preparation of crustacean chitin

Abstract: Publisher Summary This chapter describes the isolation of chitin from crustacean cuticles (shells). To isolate chitin from crustacean shells, the following three steps are required: (1) demineralization with dilute hydrochloric acid or with ethylenediaminetetraacetic acid (EDTA), (2) deproteinization with aqueous sodium hydroxide or by use of the proteolytic activity of a bacterium, and (3) elimination of lipids with organic solvent(s). The chapter tabulates composition and molecular weight of various chitin preparations from abdominal shell of Penaeus japonicus (kuruma prawn). The chapter also describes the preparation of colloidal chitin. The procedure consists of the dissolution of chitin into and the reprecipitation from concentrated hydrochloric acid and the dispersion of the reprecipitated chitin into water. Colloidal chitin serves as a substrate of chitinase and a carbon and nitrogen source of chitinolytic microorganisms.

Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli.

Abstract: Publisher Summary This chapter outlines the strategies for selecting mutants. The chapter also describes procedures to map the mutations to small regions of rRNA operons, facilitating identification of the mutations by DNA sequencing. The chapter also describes a primer extension method using rRNA templates and DNA oligonucleotide primers that permits convenient and accurate quantitation of the percent mutant rRNA in an rRNA preparation. This primer extension method is useful for determining the structural and functional capabilities of ribosomes synthesized from a single mutant rRNA operon (e.g., by analysis of total cellular ribosomes or ribosomes on polysomes) and provides a convenient way to determine if newly isolated mutations have base changes at the same position as previously isolated mutations. It also describes how a similar primer extension method can be used to accurately determine the copy number of mutant rRNA genes (e.g., plasmid copy number), permitting reliable quantitative analysis of rRNA gene expression.

[15] cAMP analog antagonists of cAMP action

Abstract: Publisher Summary The first direct evidence that (Rp)-cAMPS was a cAMP antagonist and could oppose the action of cAMP was a study done in hepatocytes in 1983. This study demonstrated that (Rp)-cAMPS is an intracellular inhibitor of cAMP action. Subsequently, this analog has been used to study several cAMPdependent systems: glucagon-induced glucose production in hepatocytes; isoproterenol-induced lipolysis in adipocytes; cAMP activation of the cellular slime mold, Dictyostelium discoideum ; cAMP-induced phosphorylation of microtubule-associated protein (MAP2) in brain tissue; and hormone-induced steroidogenesis in cultured granulosa and Leydig cells. (Rp)-cAMPS has not yet been fully exploited as an intracellular antagonist of cAMP action for several reasons: lack of availability due to difficulties involved in synthesis and purification, lack of potency, and lack of cell membrane permeability in some cell culture systems. Nevertheless, this compound, the only known intracellular antagonist of cAMP, provides a means of distinguishing cAMP-dependent from cAMP-independent cellular events, which was not previously available.-

Phylogenetic analysis using ribosomal RNA.

Abstract: Publisher Summary The inference of phylogenetic relationships from molecular data is contributing greatly in understanding the evolution of life on Earth. Although the discussion that follows is directed toward analyses based on rRNA sequences, nearly all of the concepts, and many of the details, are equally applicable to the other DNA, RNA, or protein sequences. The analysis of sequences also helps to ensure that the evolutionary inferences are based on homologous features; the interspersion of conserved with more variable sequences permits the more slowly changing sequence features to provide landmarks for identifying homologous positions in adjacent, more variable, sequences. Finally, sequence data accumulate and are constantly available for further comparisons and analyses. This contrasts with types of data that require pairwise laboratory comparisons of all the species considered. In essence, the value of a sequence increases as data from additional organisms and molecules become available. Preexisting sequences provide a framework within which new sequence data can be analyzed. The availability of sequences of homologous molecules from many species is one of the greatest assets of rRNA-based phylogenies.

[38] Experimental glomerulonephritis

Abstract: Publisher Summary This chapter discusses the analysis of experimental glomerulonephritis. There exist several animal models that closely resemble various forms of human glomerulonephritis. The resemblance includes, in most instances, morphological, immunohistological, and pathophysiological similarities. The chapter also describes different methods for producing and studying three models that cover the spectrum of fixed and planted antigens and various mediator systems. Mediators of the glomerular injury induced by such immune deposits vary widely in different models. They include the complement (C) system acting either through its terminal, membranolytic pathway or via the inflammatory action of leukocytes; antibody-directed influx of leukocytes; and antibody alone. Host species, the nature and dose of antibody, and the distribution of antigen influence which mediators will operate in the various models studied to date. The redistribution of such glomerular epithelial cell antigens follows antibody binding; subsequent shedding of the resulting complexes is thought to account for the observed subepithelial electron densities.

[2] Recognition and identification of cyanobacteria

Abstract: Publisher Summary This chapter focuses on the recognition and identification of Cyanobacteria. The recognition of cyanobacteria in the natural habitat is a prerequisite for their isolation, and, to isolate more particular members, it helps to know their ecological distribution. Following successful isolation, cyanobacteria (as other organisms) should be identified by a name, which serves as an indicator of the respective phenotypic properties and is therefore crucial for scientific communication. Unless the organisms have not been previously described, their names have to be chosen from an existing system of classification. As cyanobacteria were first recognized more than 150 years ago, a bewildering array of genera and species has been created by botanists and ecologists. Classifications were based either on the properties observable on samples collected from the natural habitat or on those extractable from dried herbarium specimens. Furthermore, many genera and species underwent repeated taxonomic revisions, leading to a large number of synonyms that only botanical experts are capable of unraveling. Cyanobacteria occupy a rather wide range of illuminated niches in terrestrial, freshwater, marine, and hypersaline environments, where they often occur in such abundance that they are readily visible by eye.

Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases

Abstract: Publisher Summary Evidence for complex formation between cellulose substantive dyes, such as Congo Red (CR), and polysaccharides has been based on changes in viscosity or solubility of polysaccharide, and on changes in spectral properties of the dye. As the formation of the complex occurs only with polymer, dye binding and the associated changes may be used to monitor hydrolytic enzyme activity. This chapter describes methods to detect complex formation and exploitation of this for detection and analysis of β-D-glucanases in solution, by means of insoluble substrate, and by gel diffusion. Use of CR binding by β-D-glucans in a gel diffusion assay was originally recommended for screening of seeds and microorganisms. The method has been applied to measurement of β-D-glucanase in commercial enzyme preparations, determination of malt (1→3)(1→4)-β-D-glucanase, and to screen and monitor purification of cellulolytic microorganisms and their enzymes. Specific examples of this application are described in this chapter.

[24] Membrane lipids

Abstract: Publisher Summary This chapter describes procedures in cyanobacterial lipid analysis, including extraction and fractionation of lipids, analysis of their fatty acids, determination of positional distribution of fatty acids within the lipids, and analysis of lipid molecular species Cyanobacterial cells contain two types of membrane, the plasma membrane and thylakoid membranes, which are distinct from each other in their composition of proteins, lipids, and pigments The composition of the fatty acids of the lipids in both types of membrane changes with growth temperature so that cyanobacterial cells adapt themselves to the environmental temperature Major lipid classes in cyanobacterial membranes are monogalactosyl diacylglycerol (MGDG), monoglucosyl diacylglycerol (GIcDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), and phosphatidylglycerol (PG) Thin-layer chromatography (TLC) is a convenient method for separating major classes of lipids The content and composition of fatty acids in lipids are determined by gas chromatographic analysis of the methyl esters which are obtained by methanolysis of the lipids Methanolysis is generally performed without isolating lipids from the silica gel