Methods for Investigation of Amino Acid and Protein Metabolism

Antoine E. El-Khoury

• Publisher: CRC Press
• Year: 2017
• ISBN:
  9781351431927
• Edition: 1
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Containing all the new as well as classical methodologies used in the investigation of amino acid and protein metabolism in human and animal models, this book is needed because of the dramatic increase in research in this field. There is no other book currently on the market that covers these methods of investigation.
Methods for Investigation of Amino Acid and Protein Metabolism explores areas such as amino acid transfer across tissue membranes, past and new applications using stable isotopes, protein synthesis in organs and tissues, and more. Because of the importance of research methods in the field of amino acid and protein nutrition and metabolism, this book facilitates the reader's integration of the concepts involved in these investigative research methods and their corollaries.
In addition to helping any nutrition investigator design and conduct appropriate research protocols in this area of nutrition, this book assists students who are planning to investigate amino acid and protein metabolism in humans or laboratory animals.

1	Investigation of Amino Acid Transfer Across Tissue Membranes Peter M. Taylor and Sylvia Y. Low

CONTENTS

1.1 Introduction: Functions of Amino Acid Transfers

1.2 Mechanisms of Amino Acid Transfer

1.2.1 Passive Diffusion

1.2.2 Carrier-Mediated Transport

1.2.3 Amino Acid Transport Systems

1.3 Kinetic Properties of Amino Acid Transporters

1.4 Techniques for Studying Amino Acid Transfers: An Overview

1.4.1 Experimental Design and Rationale

1.4.2 General Principles of Tracer Flux Methodologies

1.4.3 Analysis and Interpretation of Flux Data

1.5 Experimental Preparations

1.5.1 in vivo Techniques

1.5.1.1 Whole-Body Investigations

1.5.1.2 Regional and Tissue Studies

1.5.2 Isolated Organs And Tissues

1.5.3 Single Cells

1.5.4 Plasma Membrane Vesicles

1.5.5 Reconstitution/Overexpression of Amino Acid Transporters

Acknowledgments

References

1.1 INTRODUCTION: FUNCTIONS OF AMINO ACID TRANSFERS

Plasma membrane of cells is the principal physical barrier limiting protein and amino acid (AA) movement between different metabolic compartments in an organism. Control of these movements is now recognised to be important for overall control of whole-body protein metabolism.^1-5 AA transport processes fulfill a number of specialised but essential body functions alongside their basic role in supplying cellular AAs for protein synthesis and cell metabolism. These include absorption/reabsorption of AAs (from intestinal/renal lumen, respectively),^6-8 control of neurotransmission (re-uptake of A A transmitters from synaptic cleft),^8-10 and inter-organ exchange of carbon and nitrogen.^1,2 The most quantitatively important sites of AA transfer in the human body are likely to be skeletal muscle, the kidneys, and tissues bathed by the splanchnic circulation (notably the liver and small intestine).^1,2,11 Dietary protein is hydrolysed to small peptides and AAs within the intestinal lumen; although peptide transport represents a considerable proportion of the total amino-N uptake across the brush-border membrane, intracellular peptide hydrolysis means that the overall transepithelial movement of amino-N is almost entirely in the form of AAs.^1,2,12 In this article, we provide a brief overview of the mechanisms involved in AA movements across cell membranes before describing methods available for their study, focusing on techniques applicable to in vivo or intact tissue/organ investigations.

1.2 MECHANISMS OF AMINO ACID TRANSFER

1.2.1 PASSIVE DIFFUSION

The simplest mechanism by which AAs cross cell membranes is by passive diffusion. Diffusional fluxes of solutes are proportional to the concentration difference across a permeable barrier (e.g., lipid bilayer of the cell membrane) and the concentration gradient lies within the barrier itself.¹³ This relationship can be described quantitatively by a form of Fick’s First Law of Diffusion:

${\text{J}}_{1\to 2}=\text{D}.\text{A}.({\text{c}}_1-{\text{c}}_2)\text{/X}$

(1)

where J = net diffusional flux (mol/sec), D = diffusion coefficient of substance across the barrier (cm²/sec), A = barrier area (cm²), c = concentration of substance (mol/cm³), and x = barrier thickness (cm). The value of D is specific for both barrier and diffusing molecules. The flux J is directly proportional to this value and also to exchange area and solute concentration difference (Δc), whilst it is inversely proportional to membrane thickness. Both D and x are difficult to measure experimentally, so in practice the term permeability (P; cm/sec; where P = D/x) is generally used.¹³ Thus:

${\text{J}}_{1\to 2}=\text{P}\text{.A}.(\Delta {\text{c}}_{1\to 2})$

(2)

Passive diffusion across the lipid bilayer is favoured for small lipophilic molecules. The selective permeability of the membrane depends on the relative tendency of a given solute to dissolve in lipid/water, given by the solvent-water partition coefficient (K_p), with molecular size playing a secondary role.¹³ AAs are hydrophilic molecules with K_p values much lower than 1, and passive diffusion is usually much too slow for the required metabolic fluxes of AAs across membranes of living cells.^1,2 Cells have evolved “pores” (channels and transporters) which enable the membrane barrier to be bypassed for effective transmembrane exchange of polar solutes.^2,13 Important characteristics of these pores are:

(a) markedly greater solute flux than predicted by passive diffusion; (b) specificity for single or small structurally related group of substrates; (c) saturability (at least in theory); and (d) susceptiblility to specific inhibitors/inactivators.

1.2.2 CARRIER-MEDIATED TRANSPORT

Metabolically important AA movements across cell membranes involve transporters (or “carriers”) rather than channels (aqueous pores).^5,8,13,14 Transporters offer the required degree of substrate selectivity related to molecular interactions between solute “substrate” and binding site on the transporter protein, whereas channel selectivity is largely limited to discrimination between size and charge of potential substrates.^2,13,14 Transport mechanisms involve adsorption of solute from bulk fluid phase onto the binding site and a conformational change of the transport protein to move solute across the membrane. The binding site is alternately exposed to the two sides of the membrane during a transport cycle, in tandem with association or disassociation of the carrier-substrate complex.^13,14 The simplest carriers (facilitative transporters) act to “accelerate” (or facilitate) the process of diffusion down an (electro-) chemical gradient of solute. More complex carriers include co-transporters and counter-transporters, in which there is rigid coupling of the movement of two solutes either in the same or opposite directions.^13,14 Important factors influencing unidirectional flux through a transporter will include^13,14 the stoichiometry of a co-transport or counter-transport process, affinity of the binding site for each substrate, dependence on the membrane potential (particularly important if a net charge movement is involved), and the availability of substrates both at the cis and trans sides of the membrane (see Section 1.3). The physiological significance of coupling is that it allows the gradient of one solute to drive another solute uphill against an electrochemical gradient. Frequently, the gradient of Na⁺ is used to drive accumulation of metabolically active substrates (including AAs) into cells. Transport stoichiometry is physiologically important.^1,2,13,14 For Na⁺-coupled transport of solutes (e.g., AA), the equilibrium distribution ratio ([S]_i/[S]_o, where[S]_i/[S]_o denote intra-/extracellular concentrations) or “concentrating power” of the transporter is related to both the Na⁺- gradient (chemical + electrical) and coupling ratio (n); any increase in n markedly increases the achievable [S]_i/[S]_o.^13,14 Equilibrium[S]_i/[S]_o values are not achieved in vivo because of energy losses, transporter “slippage” and dissipation of [S] gradient by other mechanisms.^13,14 The Na⁺ gradient is generated largely by the Na⁺ pump (Na⁺+K⁺ATPase, a primary active transport process), and Na⁺-coupled transporters utilising this gradient represent secondary active transport processes.^2,13,14 Solute gradients generated by the latter process can in turn be used to generate uphill transport of a different substrate by heteroex-changing carriers, although with low concentrating power (this is termed tertiary active transport).^1,2,12 Detailed descriptions of models of a variety of transport processes can be found elsewhere.^13,14 Although we focus exclusively on the plasma membrane in this article, it should be recognised that transport across intracellular membranes such as those of the mitochondria, lysosome and the endoplasmic reticulum also represent potentially important steps for control of AA metabolism.^2,12

1.2.3 AMINO ACID TRANSPORT SYSTEMS

The pioneer...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Dedication
• Table of Contents
• Chapter 1 Investigation of Amino Acid Transfer Across Tissue Membranes
• Chapter 2 Whole-Body Protein Turnover in Humans: Past and New Applications Using Stable Isotopes
• Chapter 3 Protein Synthesis in Organs and Tissues: Quantitative Methods in Laboratory Animals
• Chapter 4 The Measurement of the Rate of Synthesis of Liver-Derived Plasma Proteins
• Chapter 5 Evaluation of the Adaptation to Protein Restriction in Humans
• Chapter 6 Determination of Amino Acid Isotopie Enrichment: Methods, Difficulties, and Calculations
• Chapter 7 The Use of GC-C-IRMS for the Analysis of Stable Isotope Enrichment in Nitrogenous Compounds
• Chapter 8 Use of Isotope Dilution Methods to Investigate Glutamine Metabolism in vivo in Humans
• Chapter 9 The Application of Muscle Biopsy in the Study of Amino Acid and Protein Metabolism
• Chapter 10 Protein and Amino Acid Metabolism in the Elderly
• Chapter 11 Evaluation of Amino Acid and Protein Metabolism in the Fetus
• Chapter 12 Analysis of Proteins in Human Milk
• Chapter 13 Studying Amino Acid and Protein Metabolism in Burn and Other Trauma Patients
• Chapter 14 Methods for Investigation of Protein and Amino Acid Metabolism in Diabetes Mellitus