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1746 results in Physiology and biological physics

Page 14 of 88

  • 23 - Enzymes and Receptors

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 809-863

  • Summary

    DEFINITION AND CLASSIFICATION OF ENZYMES

    Catalytic reactions in biological processes are facilitated by two types of catalysts, enzymes and ribozymes. Whereas enzymes are proteins, ribozymes consist of ribonucleic acid (RNA). Most enzymes are much larger than the substrates they process, but this is not a requirement (for example, restriction enzymes that cleave DNA). The catalytic features of enzymes arise through a particular three-dimensional arrangement of functional groups in a small number of amino acids (see Section 23.4.2) in the active site of the enzyme. The geometrical arrangement of such groups enables productive interactions with the bound substrate and leads to formation of a transition state for which the energy barrier (activation energy) is significantly reduced as compared to the non-catalysed reaction (see Figure 23.1). Consequently, the reaction rate is increased by several orders of magnitude relative to the non-catalysed reaction. Importantly, enzymes do not alter the position of equilibrium of the reversible reactions they catalyse, rather they accelerate establishment of the position of equilibrium for the reaction.

    Many enzymes are the key players in metabolic or signalling pathways. In these coordinated pathways, enzymes are collectively responsible for maintaining the metabolic needs of cells under varying physiological conditions (Section 23.5). Through their individual catalytic activities, they control the rate of a particular metabolic or signalling pathway. A range of regulatory mechanisms operates to allow short-, medium- and long-term changes in activity (Section 23.5.1). Therefore, the over- or under-expression of an enzyme can lead to cell dysfunction, which may manifest itself as a particular disease. Hence, enzymes have been the most important target in the development of therapeutics, and typical drugs for the treatment of pathological conditions act as inhibitors of particular enzymes (Sections 23.5.3). Other clinically relevant applications include monitoring of enzyme levels in assessment of disease states. For example, damage to the heart muscle as a result of oxygen deprivation following a heart attack results in the release of cellular enzymes into extra-cellular fluids and eventually into the blood. Such release can be monitored to aid diagnosis of the organ damage and to make a prognosis for the patient's future recovery (Section 10.3).

Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
  • 8th edition
  • Edited by Andreas Hofmann, Samuel Clokie
  • Published online:
    06 August 2018
    Print publication:
    19 April 2018
  • Bringing this best-selling textbook right up to date, the new edition uniquely integrates the theories and methods that drive the fields of biology, biotechnology and medicine, comprehensively covering both the techniques students will encounter in lab classes and those that underpin current key advances and discoveries. The contents have been updated to include both traditional and cutting-edge techniques most commonly used in current life science research. Emphasis is placed on understanding the theory behind the techniques, as well as analysis of the resulting data. New chapters cover proteomics, genomics, metabolomics, and bioinformatics, as well as data analysis and visualisation. Using accessible language to describe concepts and methods, and with a wealth of new in-text worked examples to challenge students' understanding, this textbook provides an essential guide to the key techniques used in current bioscience research.

  • 16 - Fundamentals of Bioinformatics

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 559-598

  • Summary

    INTRODUCTION

    Over the last decade, we have witnessed a massive expansion in demand for and access to low-cost high-throughput sequencing of nucleic acids, which can be predominantly attributed to the advent and establishment of so-called next-generation sequencing technologies (NGS; see Section 20.2.2). The availability and progressively decreasing costs of such technologies has been accompanied by an ever-increasing number of nucleic acid and protein sequences being deposited in public repositories and, in turn, by the need to draw biologically meaningful information or interpretations from these data. As a consequence, the discipline of bioinformatics has become instrumental in many areas of biology and, in particular, molecular biology.

    Bioinformatics can be defined as a ‘fusion’ of biology and informatics, which includes applied mathematics, computer sciences, information technology and statistics. This multi-disciplinary field of research includes two major components, one aimed at developing computational tools and algorithms to facilitate storage, analysis and manipulation of sequence data, and one aimed at applying such tools to the discovery of new biological insights on the organism(s) under consideration. Researchers involved in the field of bioinformatics comprise both algorithmor software- developers and end-users. While the main interest of the first group lies in writing sequence analysis programs and tools (programming, often called ‘ coding’), the second wishes to apply these tools to answer questions of biological relevance. Within this latter group, experienced end-users often download and maintain programs on their private personal computers or servers, analyse a number of sequences (thousands to millions) simultaneously, have a working knowledge of programming languages and are therefore skilled in the use of command-linebased software. On the other hand, occasional users mainly deal with a limited number of sequences and thus prefer the use of ‘user-friendly’, web-server-based tools, which often offer a reduced set of options and a limited capacity when compared with the corresponding downloadable software packages. This chapter intends to provide an overview of the basic methods and bioinformatics resources available for the analysis of nucleic acids and protein sequences, and is primarily addressed to occasional users. For details on bioinformatic analyses of largescale sequence datasets, such as those generated by NGS technologies, the reader is referred to Chapter 20, while programmatic or script access to programs will require more advanced programming skills, for example using the Python language (see Chapter 18).

  • 15 - Mass Spectrometric Techniques

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 535-558

  • Summary

    INTRODUCTION

    Mass spectrometry (MS) is an extremely valuable analytical technique in which the molecules in a test sample are converted to gaseous ions that are subsequently separated in a mass analyser according to their mass-to-charge (m / z) ratio and then detected. The mass spectrum is a plot of the relative abundances of the ions at each m / z ratio. Note that it is the mass-to-charge ratio (m / z), and not the actual mass, that is measured. For example, if a biomolecule is ionised in positive ion mode, the instrument measures the m / z after the addition of one proton (i.e. 1.0072772984 Da for exact mass or 1.0078 Da for average mass). Similarly, for a biomolecule ionised in negative ion mode, an m / z after the loss of one proton is measured.

    The essential features of all mass spectrometers are:

  • • G eneration of ions in the gas phase

  • • S eparation of ions in a mass analyser

  • • D etection of each species of a particular m / z ratio.

    • Several techniques exist to generate ions and are discussed below. Of these, the development of electrospray ionisation (ESI; Section 15.2.4) and matrix-assisted laser desorption ionisation (MALDI; Section 15.2.5), has effectively expanded the detectable mass range, enabling the measurement of almost any biomolecule. Mass analysers separate ions by use of either a magnetic or an electric field (Section 15.3); detectors produce a measurable signal in the form of either a voltage or a current that can be transformed by a computer into data to be analysed (Section 15.4). The symbol M r is used to designate relative molecular mass. As a relative measure, M r has no units.

    The treatment of mass spectrometry in this chapter will be rather non-mathematical and non-technical. Mass spectrometry has a wide array of applications, including drug discovery and the sciences of proteomics (Chapter 21) and metabolomics (Chapter 22).

    This chapter will focus on the fundamental principles of mass spectrometry. The intention is to give an overview of the different types of instrumentation that are available and discussion of their applications, complementary techniques and the advantages/disadvantages of each system. Sample preparation and data analysis will also be covered. A further reading list is provided, covering more technical and mathematical aspects of mass spectrometry.

  • Foreword to the Eighth Edition

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp xiii-xiv

  • Summary

    When the first edition of our book was published in 1975, biochemistry was emerging as a new discipline that had the potential to unify the previously divergent -ologies that constituted the biological sciences. It placed emphasis on the understanding of the structure, function and expression of individual proteins and their related genes, and relied on the application of analytical techniques such as electrophoresis, chromatography and various forms of spectrometry. In the ensuing 40 years the completion of the Human Genome Project has confirmed the central role of DNA in all of the activities of individual cells and the emergence of molecular biology as the means of understanding complex biological processes. This in turn has led to the new disciplines of bioinformatics, chemoinformatics, proteomics and metabolomics. The succeeding six editions of our book have attempted to reflect this evolution of biochemistry as a unifying discipline. All editions have placed emphasis on the experimental techniques that undergraduates can expect to encounter during the course of their university studies and to this end we have been grateful for the excellent feedback we have received from the users of the book.

    The point has now been reached where we believed that it was appropriate for us to hand over the direction and academic balance of future editions to a new editorial team and to this end we are delighted that Andreas Hofmann and Samuel Clokie have agreed to take on the role. We wish them well and look forward to the continuing success of the book.

  • 12 - Centrifugation and Ultracentrifugation

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 424-453

  • Summary

    INTRODUCTION

    Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. The smaller the particles, the higher the g-forces (see next section) required for the separation. It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and, with higher g-force instruments or ‘ultra’-centrifuges (up to 60 000 revolutions per minute corresponding to ~ 200 000×g) isolated macromolecules such as proteins or nucleic acids. Such high-speed devices require a vacuum to avoid overheating of samples. The development of the first analytical ultracentrifuge – with a specially designed optical system for monitoring and recording the sedimentation process – by Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades. Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all invasive subcellular studies. While analytical ultracentrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies, preparative centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest.

    Most undergraduate students will be exposed to preparative centrifugation protocols during practical classes and might also experience a demonstration of analytical centrifugation techniques. This chapter is accordingly divided into a short introduction into the theoretical background of sedimentation, an overview of practical aspects of using centrifuges in the biochemical laboratory, an outline of preparative centrifugation and a description of the usefulness of ultracentrifugation techniques in the biochemical characterisation of macromolecules. To aid in the understanding of the basic principles of centrifugation, the general designs of various rotors and separation processes are diagrammatically represented. Often, the learning process of undergraduate students is hampered by the lack of a proper linkage between theoretical knowledge and practical applications. To overcome this problem, the description of preparative centrifugation techniques is accompanied by an explanatory flow chart and the detailed discussion of the subcellular fractionation protocol for a specific tissue preparation.

  • 9 - Radioisotope Techniques

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 313-345

  • Summary

    WHY USE A RADIOISOTOPE?

    When researchers contemplate using a radioactive compound there are several things they have to consider. First and foremost, they must ask the question: is a radioisotope necessary or is there another way to achieve the objectives? The reason for this is that use of radioisotopes is governed by very strict legislation. The rules are based on the premise that radioactivity is potentially unsafe (if handled incorrectly) and should therefore only be used if there are no alternatives. Then, once it is decided that there is no alternative, the safest way of carrying out the work needs to be planned. Essentially this means using the safest isotope and the smallest amount possible. But why do we use radioisotopes in the first place? First, it is possible to detect radioactivity with exquisite sensitivity. This means that, for example, the progress of an element through a metabolic pathway or in the body of a plant or animal can be followed relatively easily. Very small amounts of a radioactive molecule are needed, and detection methods are well established. Second, it is possible to follow what happens in time. Imagine a metabolic pathway such as carbon dioxide fixation (the Calvin cycle). All the metabolites in the cycle are present simultaneously; so a good way to establish the order of the metabolism is to add a radioactive molecule (in this example, 14 C-labelled carbon dioxide in the form of sodium bicarbonate) and see what happens to it by extracting the metabolites from the plant and identifying the radioactive ones. Third, it is possible to trace what happens to individual atoms in a pathway. This is done, for example, by creating compounds with a particular isotope in specific locations in the molecule. Fourth, we can identify a part or end of a molecule, and follow reactions very precisely. This has been very useful in molecular biology, where it is often necessary to label one end of a DNA molecule (e.g. for techniques such as gel mobility shift assay or DNA footprinting, methods for investigating sequence-specific DNA protein binding), or immunochemical diagnostics; see Section 7.2.9).

  • H1 - Electron Microscopy

  • from Part H - ELECTRON DIFFRACTION
  • Nathan R. Zaccai, University of Cambridge, Igor N. Serdyuk, Joseph Zaccai
  • Book:
    Methods in Molecular Biophysics
    Published online:
    28 May 2018
    Print publication:
    18 May 2017, pp 489-501

  • Summary

    HISTORICAL REVIEW

    1820

    J.-B. Biot and F. Savart collaborated on the theory of magnetism. Their eponymous law (formulated in 1820) describes the motion of a charge in a magnetic field.

    1872

    Ernst Abbe formulated his wave theory of microscopic imaging. In 1878, he was able to correlate resolution and wavelength of light mathematically. A few years later, through the company Zeiss, he commercialized the first ever lenses to have been designed based on sound optical theory and the laws of physics.

    1873

    In the second half of the nineteenth century, J. C. Maxwell established the laws of electromagnetism. His equations first appeared in fully developed form in his book Electricity and Magnetism (1873).

    1897

    J. J. Thomson discovered electrons by demonstrating cathode rays were actually units of electrical current made up of negatively charged particles of subatomic size. For these investigations, he won the Nobel Prize for physics in 1906.

    1924

    In his doctoral thesis, de Broglie theorized that matter has the properties of both particles and waves. He received the 1929 Nobel Prize for physics. The particle–wave duality was confirmed experimentally for the electron a few years later by C. J. Davisson and L. H. Germer, and by G. P. Thomson. Thomson and Davisson jointly shared the Nobel Prize in physics in 1937.

    1926

    H. Busch described a lens to focus electrons. H. Ruska subsequently built the first working electron microscope in 1930 (Nobel Prize in 1986). Ruska's 1931 paper contains for the first time the term “electron microscopy.” The principle behind this type of microscope has not varied significantly since.

    1932

    Fritz Zernike invented the phase-contrast light microscope, which uses a quarter-wave plate. He was awarded the 1953 Nobel Prize for physics for this work, which opened up the study of transparent biological materials. The feasibility of a scanning electron microscope was demonstrated by Knoll in 1935, and the first prototype was built by M. Van Ardenne three years later. By the late 1930s, electron micrographs of recognizable value to biologists were being published. In 1942, S. E. Luria obtained the first high-quality electron micrograph of a bacteriophage.

    1940s

    The negative staining technique came into extensive use in the 1940s, although it was only in 1955 that C. E. Hall recognized, and deliberately used, phosphotungstic acid as a negative stain. In 1959, S. J. Singer used ferritin-coupled antibodies to detect cellular molecules.

  • B1 - Mass and Charge

  • from Part B - MASS SPECTROMETRY
  • Nathan R. Zaccai, University of Cambridge, Igor N. Serdyuk, Joseph Zaccai
  • Book:
    Methods in Molecular Biophysics
    Published online:
    28 May 2018
    Print publication:
    18 May 2017, pp 71-84

  • Summary

    HISTORICAL REVIEW

    1897

    J. J. Thomson made the first measurement of the mass-to-charge ratio of elementary particle “corpuscles,” which later became known as electrons. This can fairly be considered as the birth of mass spectrometry.

    1918–1919

    A. Dempster and F. Aston developed the first mass spectrographs. Photographic plate was used as the array detector. The instruments were used for isotopic relative abundance measurements.

    1951

    W. Pauli and H. Steinwedel described the development of a quadrupole mass spectrometer. The application of superimposed radio-frequency and constant potentials between four parallel rods acted as a mass separator in which only ions within a particular mass range perform oscillations of constant amplitude and are collected at the far end of the analyzer.

    1959

    K. Biemann was the first to apply electron ionization mass spectrometry to the analysis of peptides. Later it was shown that for sequence determination, peptides had to be derivatized prior to analysis by a direct probe.

    1968–1970

    M. Dole was the first to bring synthetic and natural polymers into the gas phase at atmospheric pressure. This was done by spraying a sample solution from a small tube into a strong electric field in the presence of a flow of warm nitrogen, to assist desolvation. First experiments on lysozyme demonstrated the phenomenon of multiple charging.

    1974

    D. Torgerson introduced plasma desorption mass spectrometry. This technique uses 252Cf fission fragments to desorb large molecules from a target. It was the first of the particle-induced desorption methods to demonstrate that gas-phase molecular ions of proteins could be produced from a solid matrix.

    1974

    B. Mamyrin made the most important contribution to the development of time-of-flight (TOF) mass spectrometry. He constructed the so-called reflectron device, which had been proposed by S. Alikanov in 1957. The reflectron essentially improves mass resolution in the TOF mass spectrometer.

    1978

    N. Commisarow and A. Marshall adapted Fourier transform methods to ion cyclotron resonance spectrometry and built the first Fourier transform mass instrument. Since that time, interest in this technique has increased exponentially, as has the number of instruments.

  • A1 - Macromolecules in Their Environment

  • from Part A - BIOLOGICAL MACROMOLECULES AND PHYSICAL TOOLS
  • Nathan R. Zaccai, University of Cambridge, Igor N. Serdyuk, Joseph Zaccai
  • Book:
    Methods in Molecular Biophysics
    Published online:
    28 May 2018
    Print publication:
    18 May 2017, pp 13-24

  • Summary

    HISTORICAL REVIEW

    The discovery of biological macromolecules is tightly interwoven with the history of physical chemistry, which formally emerged as a discipline in 1887, when the journal founded by Jacobus Van't Hoff and Wilhelm Ostwald, Zeitschrift für Physikalische Chemie, was first published. Interestingly, the first papers were concerned with reactions in solution, because biological processes essentially take place in the aqueous environment inside living cells.

    The nineteenth-century discoveries of solution properties that led to our knowledge about biological macromolecules are described briefly in the Introduction. We must also mention the Grenoble chemist François-Marie Raoult (1886), who formulated the freezing-point depression law that made it possible to determine the molecular weight of dissolved substances, and Hans Hofmeister (1895), a medical doctor and physiologist who was interested in the diuretic and laxative effects of salts, and classified them according to how they modified the solubility of protein in aqueous solutions. The Hofmeister series was later established as a ranking order of the “salting-out,” or precipitating, efficiency of ions. Gilbert Newton Lewis introduced the concepts of activity in 1908, and of ionic strength, with Merle Randall in 1921. In 1911, Frederick George Donnan published a paper on the membrane potential developed during dialysis of a non-permeating electrolyte. Peter Debye and Erich Hückel (1923) proposed a theory for electrolyte solutions. In recent decades, modern methods, such as dynamic light scattering and small-angle neutron scattering, developed for the characterization of polymers, and especially polyelectrolytes, have contributed significantly to our current understanding of biological macromolecules in solution.

    There is now a growing interest in the behavior of proteins in non-aqueous solvents and even in vacuum, mainly in the context of biotechnology, but also with respect to whether or not water is essential to life. Proteins, as active biological particles, have evolved in the presence of water, however, and, to a large extent, cannot be considered separately from their aqueous environment. We recall that even crystals of biological macromolecules contain an appreciable amount of solvent and should be considered as organized macromolecular solutions.

Page 14 of 88