Membrane Analysis

Dr John Graham, Joan Higgins

• Publisher: Garland Science
• Year: 2020
• Print ISBN: 9781872748887
• eBook ISBN: 9781000141023
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Membrane Analysis provides a comprehensive review of laboratory methods for membrane study, with an emphasis on isolating membranes, analysing their composition and architecture, and investigating membrane function.

1 General membrane composition and structure

All membranes have an amphipathic nature, i.e. they have a hydrophobic (non-polar) central layer sandwiched between two hydrophilic (polar) surfaces, and the molecules and macromolecules which membranes contain are designed to conform to and maintain this basic structure. The basic unit has a thickness of 5–8 nm. There are three principal components of membranes, lipid, protein and carbohydrate.

1.1 Lipids

1.1.1 General lipid composition

Membranes are composed of a bilayer of amphipathic lipids: although there is evidence from physical studies (NMR spectroscopy and X-ray diffraction) that nonbilayer structures (e.g. hexagonal arrays or micelles) are present, the bilayer structure is the predominant one.

The lipid bilayer:

• is permeable to small nonionized molecules, such as glycerol, urea, water, oxygen and carbon dioxide and to all lipophilic molecules;

• provides a structural framework and the proper environment for the functioning of many membrane proteins.

The total amount of lipid in a membrane is normally expressed as a protein:lipid weight ratio (see Table 1.1). Generally this ratio is >1.0 and in the most metabolically active membranes may approach 4.0. Eukaryotic surface membranes, however, tend to have a lower ratio (i.e. more lipid) than do membranes of cytoplasmic organelles or the prokaryotic cytoplasmic membrane, although only in lipid-rich myelin does the weight ratio fall significantly below 1.0.

Table 1.1. Protein:lipid ratios in membranes

Membrane	Protein:lipid	Membrane	Protein:lipid
Animal
Myelin	0.25	Erythrocyte	1.1
Surface (liver)	1.5	Nuclear (liver)	2.0
Rough ER (liver)	2.5	Smooth ER (liver)	2.1
Inner mit. (liver)	3.6	Outer mit. (liver)	1.2
Golgi (liver)	2.4	Sarcoplasmic reticulum	3.0
Retinal rod	1.0
Plant
Surface	0.9	Chloroplast	1.9
Saccharomyces
Surface	1.2
Prokaryote surface
Bacillus	2.8	Micrococcus	2.4
Staphylococcus	2.4	Escherichia coli	2.8
Outer membrane of Gram-negative bacteria	2.2

Because protein is much denser than lipid, membranes with different protein:lipid ratios may also have different densities – one of the properties that is used to fractionate membranes by centrifugation (see Chapter 3).

Generally lipids can be divided into four major classes:

• free fatty acids;

• esters of fatty acids, e.g. triacylglycerol and phospholipids;

• isoprenoids, e.g. sterols, sterol esters, dolichol and farnesol;

• glycolipids.

Phospholipids are major components of all membranes and, with the exception of myelin, they represent greater than 50% of the total membrane lipid mass. Glycolipids and cholesterol are usually concentrated in the plasma membrane of mammalian cells, while in plants only sterols (mostly stigmasterol and sitosterol) show this preferential localization and glycolipids predominate in the chloroplast. Bacterial membranes may also be rich in glycolipid but, with the exception of mycoplasmas, they lack sterols, and even in mycoplasmas the sterol is derived almost exclusively from the growth medium. It is only relatively recently that isoprenoids other than sterols (dolichol phosphate and derivatives of farnesol and geranylgeranol) have been shown to be important in membranes. The relative amounts of glycolipid, phospholipid and cholesterol in some examples of membrane types is given in Table 1.2.

Table 1.2. Lipid composition of membranes (figures are % of total lipid mass)

Membrane	Phospholipid	Glycolipid	Sterol
M...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Abbreviations
• Preface
• 1. General membrane composition and structure
• 2. The membranes of prokaryotes and eukaryotes
• 3. Preparation of subcellular membranes
• 4. Molecular composition
• 5. Investigating the topology of membrane proteins
• 6. Investigation of membrane lipids
• 7. Protein targetting and membrane biogenesis
• 8. Endocytosis and vesicular trafficking
• Index