Are receptors integral membrane proteins or peripheral membrane proteins?

Integral membranes proteins serve as transporters. Peripheral proteins serve as cell adhesion molecules, antigens and enzymes. So what about receptors? Which proteins carry out the duty of receptors?

All the receptors I know about are integral, transmembrane proteins. It would certainly be possible for a receptor domain to exist on a peripheral protein that interacted with a transmembrane protein, but I don't know of any examples of this: an extracellular peripheral protein would tend to float away, which is a bit of a problem if they aren't intended for secretion.

There are certainly peripheral proteins that associate with the internal parts of transmembrane receptor proteins and may influence their function, but they are not themselves receptors.

I'm not sure about your question about "which proteins" - those proteins are most often named after their ligand followed by the word "receptor", or based on their function that is triggered by binding their ligand, or both (i.e., the AMPA receptor is a glutamate receptor that is also activated by AMPA; receptor tyrosine kinases are a family of receptors that are tyrosine kinases; the individual members of the family are typically known by their ligand).

Lipid-anchored protein

Lipid-anchored proteins (also known as lipid-linked proteins) are proteins located on the surface of the cell membrane that are covalently attached to lipids embedded within the cell membrane. These proteins insert and assume a place in the bilayer structure of the membrane alongside the similar fatty acid tails. The lipid-anchored protein can be located on either side of the cell membrane. Thus, the lipid serves to anchor the protein to the cell membrane. [1] [2] They are a type of proteolipids.

The lipid groups play a role in protein interaction and can contribute to the function of the protein to which it is attached. [2] Furthermore, the lipid serves as a mediator of membrane associations or as a determinant for specific protein-protein interactions. [3] For example, lipid groups can play an important role in increasing molecular hydrophobicity. This allows for the interaction of proteins with cellular membranes and protein domains. [4] In a dynamic role, lipidation can sequester a protein away from its substrate to inactivate the protein and then activate it by substrate presentation.

Overall, there are three main types of lipid-anchored proteins which include prenylated proteins, fatty acylated proteins and glycosylphosphatidylinositol-linked proteins (GPI). [2] [5] A protein can have multiple lipid groups covalently attached to it, but the site where the lipid binds to the protein depends both on the lipid group and protein. [2]

Structure of Peripheral Proteins

In the image below, several peripheral proteins are labeled. A peripheral protein does not have a definite structure, but it has several key aspects which make it a peripheral protein.

First, all peripheral proteins are associated with the cell membrane. The amino acid sequences of these proteins are unique in that they draw the proteins to the membrane, and they tend to congregate on the surface of the membrane. This allows them to be in the right place to carry out their designated action. In the image, the orange peripheral proteins are seen attached to either the phosphoglyceride lipid molecules which make up the lipid bilayer, or to integral proteins. A protein without these areas of amino acids would not be attracted to the membrane. It would be distributed evenly throughout the cytoplasm, and would not be a peripheral protein.

Second, peripheral proteins do not have a hydrophobic region of amino acids. This, and the polarity of other amino acid groups, keeps the peripheral proteins on the surface of the cell membrane. This is due to the amphipathic nature of phosphoglycerides. This means that the blue “head” region is polar and hydrophilic. The yellow “tails”, which constitute the middle of the membrane, are hydrophobic. To avoid being sucked into the membrane, peripheral proteins often have lots of hydrophilic amino acids exposed on their surface. Integral proteins expose hydrophobic amino acids in the middle, and hydrophilic amino acids on the parts exposed to water. This effectively locks them within the membrane.

The plasma membrane contains molecules other than phospholipids, primarily other lipids and proteins. The green molecules in the figure below, for example, are the lipid cholesterol. Molecules of cholesterol help the plasma membrane keep its shape. Many of the proteins in the plasma membrane assist other substances in crossing the membrane.

The plasma membranes also contain certain types of proteins. A membrane protein is a protein molecule that is attached to, or associated with, the membrane of a cell or an organelle. Membrane proteins can be put into two groups based on how the protein is associated with the membrane.

Integral membrane proteins are permanently embedded within the plasma membrane. They have a range of important functions. Such functions include channeling or transporting molecules across the membrane. Other integral proteins act as cell receptors. Integral membrane proteins can be classified according to their relationship with the bilayer:

  • Transmembrane proteins span the entire plasma membrane. Transmembrane proteins are found in all types of biological membranes.
  • Integral monotopic proteins are permanently attached to the membrane from only one side.

Some integral membrane proteins are responsible for cell adhesion (sticking of a cell to another cell or surface). On the outside of cell membranes and attached to some of the proteins are carbohydrate chains that act as labels that identify the cell type. Shown in the figure below are two different types of membrane proteins and associated molecules.

Peripheral membrane proteins are proteins that are only temporarily associated with the membrane. They can be easily removed, which allows them to be involved in cell signaling. Peripheral proteins can also be attached to integral membrane proteins, or they can stick into a small portion of the lipid bilayer by themselves. Peripheral membrane proteins are often associated with ion channels and transmembrane receptors. Most peripheral membrane proteins are hydrophilic.

Some of the membrane proteins make up a major transport system that moves molecules and ions through the polar phospholipid bilayer.

The Fluid Mosaic Model

In 1972, S.J. Singer and G.L. Nicolson proposed the now widely accepted Fluid Mosaic Model of the structure of cell membranes. The model proposes that integral membrane proteins are embedded in the phospholipid bilayer, as seen in the figure above. Some of these proteins extend all the way through the bilayer, and some only partially across it. These membrane proteins act as transport proteins and receptors proteins.

Their model also proposed that the membrane behaves like a fluid, rather than a solid. The proteins and lipids of the membrane move around the membrane, much like buoys in water. Such movement causes a constant change in the “mosaic pattern” of the plasma membrane.

Extensions of the Plasma Membrane

The plasma membrane may have extensions, such as whip-like flagella or brush-like cilia. In single-celled organisms, like those shown in the figure below, the membrane extensions may help the organisms move. In multicellular organisms, the extensions have other functions. For example, the cilia on human lung cells sweep foreign particles and mucus toward the mouth and nose.

Flagella and Cilia. Cilia and flagella are extensions of the plasma membrane of many cells.

Difference Between Integral Proteins and Peripheral Proteins

Proteins are considered as macro molecules, which consist of one or more polypeptide chains. The polypeptide chains are made up of amino acids bonded together by peptide bonds. The primary structure of a protein can be determined by the amino acid sequence. Certain genes code for many proteins. These genes determine the sequence of amino acid, thereby determining their primary structure. Integral and peripheral proteins are considered as ‘plasma membrane proteins’ due to their occurrence. These proteins are generally responsible for a cell’s ability to interact with the external environment.

Integral Protein

Integral proteins are mainly found either fully or partially submerged in the phospholipids bilayer of the plasma membrane. These proteins have both polar and non-polar regions on them. Polar heads protrude from the surface of the bilayer while non-polar regions are embedded in it. Usually only the non-polar regions interact with the hydrophobic core of the plasma membrane by making hydrophobic bonds with the fatty acid tails of the phospholipids.

The integral proteins that span the entire membrane from the inner surface to the outer surface are called transmembrane proteins. In transmembrane proteins, both ends that project out of the lipid layer are polar or hydrophilic regions. The middle regions are non-polar and have hydrophobic amino acids on their surface. Three types of interactions help to embed these proteins in the lipid bilayer, namely, ionic interactions with the polar heads of phospholipid molecules, hydrophobic interactions with the hydrophobic tails of phospholipid molecules and specific interactions with certain regions of lipids, glycolipids or oligosaccharides.

Peripheral Protein

Peripheral proteins (extrinsic proteins) are present on the innermost and outermost of phospholipids bilayer. These proteins are loosely bound to the plasma membrane either directly by interactions with polar heads of phospholipids bilayer or indirectly by interactions with integral proteins. These proteins constitute about 20-30 % of total membrane proteins.

Most of the peripheral proteins are found on the innermost surface or cytoplasmic surface of the membrane. These proteins remain bounded by either through covalent bonds with fatty chains or through an oligosaccharide to phospholipids.

What is the difference between Integral and Peripheral Protein?

• Peripheral proteins occur on the surface of plasma membrane whereas integral proteins occur either fully or partially submerged in the lipid layer of plasma membrane.

• Peripheral proteins are loosely bound to the lipid bilayer and do not interact with the hydrophobic core in between two layers of phospholipids. In contrast, integral proteins are tightly bound and are directly interacting with the hydrophobic core of the plasma membrane. Due to these reasons, integral protein dissociation is more difficult than peripheral proteins.

• Mild treatments can be used to isolate peripheral proteins from the plasma membrane, but for isolation of integral proteins, mild treatments are not enough. To break the hydrophobic bonds, detergents are required. Thus, integral proteins can be isolated from the plasma membrane.

• After isolation of these two proteins from the plasma membrane, peripheral proteins can be dissolved in neutral aqueous buffers while integral proteins cannot be dissolved in neutral aqueous buffers or aggregates.

• Unlike peripheral proteins, integral proteins are associated with lipid when solubilized.

• Examples of peripheral proteins are spectrin of erythrocytes, cytochrome C and ATP-ase of mitochondria and acetylcholinesterase in electroplax membranes. Examples of integral proteins are membrane bounded enzymes, drug and hormone receptors, antigen and rhodopsin.

• Integral proteins represent around 70% while peripheral proteins represent the remaining portion of plasma membrane proteins.

What Is an Integral Membrane Protein? (with pictures)

An integral membrane protein, also known as an IMP, is one which spans the entire biological membrane of a cell. These proteins are attached permanently to the cell membrane, and their function typically relies on being present in the membrane. Both structurally and functionally, they are integral parts of the membranes of cells.

Each integral membrane protein molecule has an intricate relationship with the membrane within which it is situated. Structurally, the IMP is usually placed such that protein strands are woven throughout the structure of the cell membrane. Sections of protein protrude through the cell wall inside or outside the cell, or in both directions. The protein molecule cannot function if it is not embedded within the membrane.

Another feature of the protein is that these proteins can be removed from the membrane only with very specific chemical treatment. This is because hydrophobic regions of the protein are protected within the phospholipid bilayer of the cell membrane. For this reason, detergents, denaturing solvents, and nonpolar solvents must be used to disrupt the phospholipid bilayer and extract the integral membrane protein.

Within the integral membrane protein class are several different categories of protein, many of which are receptors and other types of cell signaling molecules. They are categorized into two groups, based on their structure. These are integral transmembrane proteins, and integral monotopic proteins.

Integral transmembrane proteins are those which span the entire cell membrane. These proteins may span the membrane once, or may span it several times, weaving through the phospholipid bilayer such that there are several pieces of the protein protruding through the cell wall. Overall this is the most common type of IMP.

Examples of integral transmembrane proteins include voltage-gated ion channels such as those which transport potassium ions in and out of cells. Certain types of T cell receptors, the insulin receptor, and many other receptors and neurotransmitters, are all integral transmembrane proteins. In general, receptors, transmitters, and transporters tend to belong to this class of IMP because proteins that span the entire membrane are typically able to sense conditions both inside and outside of the cell simultaneously.

Integral monotopic proteins do not span the entire biological membrane. Instead they are attached to the membrane from only one side, with one end of the protein protruding either inside or outside the cell. This class of proteins includes enzymes such as monoamine oxidase and fatty acid amide hydrolase. Integral monotopic proteins are unable to sense conditions both inside and outside the cell, and are less likely to be involved in intercellular signaling.

Membrane Proteins Extraction

Membrane proteins (MPs), part of biological membranes, play a crucial role in basic cellular structure and functions, including cell integrity, signal transduction, molecular recognition, material transport, and cell-to-cell communication. Additionally, MPs are the largest category of drug targets. More than 60% currently available therapeutic molecules target one or more MPs. However, there are relatively few MPs with known crystal structures due to the technical challenges associated with membrane protein extraction, solubilisation, and purification.

MPs are divided into two major classes: integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs). IMPs are permanently attached to the membrane lipid bilayers. While, PMPs are temporarily associated with either the lipid bilayer or IMPs by means of non-covalent interactions. Generally speaking, IMPs can be purified by more stringent techniques than PMPs, whose extraction just needs a high PH buffer. Here we provide a protocol for IMPs extraction focusing on homogenate preparation, soluble proteins removal, membrane proteins extraction and detergent removal.

  • PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH to 7.4.
  • Homogenate buffer: 250 mM sucrose, 1 mM EDTA, 10 mM Tris-HCl buffer, pH to 7.2, add protease and phosphatase inhibitors cocktail before use.
  • Blocking buffer: 0.1% (w/v) bovine serum albumin in 50 mM Tris–HCl, 0.15 M NaCl, pH 7.4.
  • Washing buffer: 50 mM Tris–HCl, 0.15 M NaCl, pH 7.4.
  • 2% Triton X-100
  • Scissors and scalpel blade
  • Pipettes and pipettors
  • Homogenizer
  • Microcentrifuge tube
  • Sonication
  • Centrifuge
  • Columns and appropriate detergent absorption matrix

Homogenate preparation

1. For cultured cells: collect cells (0.2-1 x 10 8 ) and wash with ice-cold PBS. Centrifuge at 500 x g for 5 min at 4°C and aspirate the PBS. Resuspend the pellet in 2 mL ice-cold homogenization buffer in a mechanical homogenizer. Homogenize cells on ice. Then skip to Step 3.

2. For tissues: weigh a certain amount of tissues and mince into pieces on ice. Wash with PBS, centrifuge at 500 x g for 5 min at 4°C and discard the wash buffer. Homogenize tissues on ice in 2 volumes of the homogenize buffer, until it is completely lysed.

3. (Optional) Sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator. Keep sample in an ice bath and keep probe away from the sample-air interface to minimize foaming.

Soluble proteins removal

4. Transfer the homogenate to a fresh 1.5 mL Ep tube and centrifuge at 700 x g for 10 min at 4°C to remove the intact cells, nuclei and cell debris.

5. Collect the supernatant and discard the pellet.

6. Centrifuge the supernatant at 100,000 x g for one hour at 4°C.

7. Carefully aspirate the supernatant (containing cytosol fraction) and collect the pellet.

8. Wash the pellet with homogenization buffer and re-centrifuge at 100,000 x g for one hour at 4°C. Collect the pellet.

Membrane proteins extraction by Triton X-100

9. Resuspend the pellet in 1 mL homogenization buffer.

10. Add cells drop-wise to the 2% Triton X-100 while stirring

11. Incubate for 30 min at 4°C with occasional vortexing.

12. Centrifuge at 100,000×g for 30 min at 4°C.

13. Transfer the supernatant to a fresh tube.

Note: For peripheral membrane proteins fractionation, you just need resuspend the pellet (obtained in Step 8) in high pH buffer (100 mM Na2CO3, pH 11.3). Incubate for 30 min at 4°C with occasional vortexing and centrifuge at 100,000 x g for one hour at 4°C. Collect the supernatant.

Detergent removal by adsorption chromatography

Note: Before starting, ensure that your detergent is non-ionic detergent (e.g. Triton X-100) and the molecular weight of protein is large enough to avoid entrapment in the pores of the absorption matrix.

14. Apply distilled water through the column matrix, followed by blocking buffer.

Membrane Protein Platform

Creative Biolabs has established custom membrane protein and membrane protein antibody production platforms for antibody discovery.

Membrane proteins, actually, are a kind of proteins acting as ion channels, receptors and transporters, which enable the cells to transport environmental signals across the biological membranes. Membrane proteins can be classified into two categories, integral (intrinsic) and peripheral (extrinsic) membrane proteins—based on the nature of the membrane-protein interactions (Figure 1).

Figure 1. Schematic of membrane proteins in biological membrane. (Molecular cell biology, 4 th edition)

Integral membrane proteins, have one or more segments that are embedded in the phospholipid bilayer, of which most span the entire phospholipid bilayer. These transmembrane proteins contain one or more hydrophobic membrane-spanning domains (α helices or multiple β strands), extending into the aqueous environment. Peripheral membrane proteins, do not interact with the hydrophobic core of the phospholipid bilayer. Instead they are commonly bound to the plasma membrane by indirect interactions with integral membrane proteins or by dirct interactions with lipid polar head groups. Peripheral proteins are located to the cytosolic face of the plasma membrane, playing a role in signal transduction. Other peripheral proteins, including certain proteins of the extracellular matrix, are localized to the outer (exoplasmic) surface of the plasma membrane.

It has been proven that membrane proteins perform a wide range of functions in cell growth, cell–cell communication (signaling transduction), differentiation, flow of information, metabolism, and migration. Defects in some membrane proteins can lead to diseases, such as cancer, therefore, membrane proteins represent nearly 50% of the targets of therapeutic research. It is very difficult to study membrane proteins due to their naturally low expression levels, although, the study of the structures of membrane proteins is a hot-spot issue all over the world because this will help to better understand the functions of these membrane proteins. Creative Biolabs has many unparalleled expression systems to obtain high-yield and native conformational membrane proteins.

Except for membrane protein production, Creative Biolabs can provide customized membrane protein antibody production services. Solubilization of the membrane protein immunogens with detergents may lead to major conformational changes, making their testing with monoclonal antibodies by immune blot and ordinary immunoprecipitation difficult. Using unique MPAT™ and MEAD™ technoligies, Creative Biolabs is specialized in membrane protein and its antibody production services. Please feel free to contact us for a detailed quote.

  1. A. M. Seddon, et al. (2004). Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophy. Acta., 1666(1-2): 105-117.
  2. Membrane protein. (
  3. Membrane proteins. Molecular Cell Biology. 4th edition. Section 3.4.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

1.2 Membrane Features

The native environment of membrane proteins is dynamic and asymmetric, described by Singer and Nicolson as fluid and mosaic in their 1972 model that became the paradigm for membrane structure (Figure 1.1). 4 Lipids consisting of polar head groups and nonpolar acyl chains form a two-dimensional fluid bilayer (Figure 1.2). Lipid composition is diverse, with most of the lipids randomly distributed in the bulk phase of the bilayer while some are localized by specific interactions with proteins and/or other lipids, often in regions of ordered lipids called rafts (see below).

In the lipid disordered phase (Ld, also called Lα), fluidity of the bilayer results from the constant and varied motion of lipids (Figure 1.3). Although the lateral diffusion rate in pure lipid bilayers is very fast, the measured mobility of bulk lipids on the surface of cells is much slower. This difference has been explained by single-particle tracking on the surface of cells containing cytoskeletons: single-molecule trajectories are fast within small regions, where they are confined until they hop to a contiguous region, producing a slower overall progression. 5

The mosaic distribution of membrane proteins results from wide variations in lateral mobility, from those that diffuse rapidly on the surface to those anchored by the cytoskeleton. A large proportion of membrane proteins function in protein assemblies, which themselves have varying lifetimes. Some complexes of membrane proteins are very stable, such as the respiratory complexes involved in energy transduction (see below), while others are the result of transient interactions such as those involved in signal transduction. Many lipid-anchored proteins are observed in membrane rafts enriched in sphingomyelin and cholesterol in the lipid-ordered (Lo) state (see Figure 1.2). The presence of rafts varying in size (diameters from 10 to 200 nm) and in duration (from <1 ms to fairly stable lifetimes) enhances nonrandom distributions and dynamic interactions in the membrane. 6 Lipid–lipid interactions probably drive raft formation, given that even simple lipid mixtures reveal fluid immiscibility (Figure 1.4). The fused hydrocarbon rings of cholesterol are nearly rigid, allowing the sterol to align with lipids containing saturated acyl chains, especially sphingomyelins, and promote the tight packing of the Lo phase.

6 Important Types of Membrane Proteins (With Diagram)

Some of the most important types of membrane proteins are as follows:

1. Peripheral (Extrinsic) Proteins 2. Integral (Intrinsic) Proteins 3. Asymmetric Distribution of Membrane Proteins 4. Mobility of Membrane Proteins 5. Enzymatic Properties of Membrane Proteins 6. Isolation and Characterization of Membrane Proteins.

1. Peripheral (Extrinsic) Proteins:

Peripheral or extrinsic membrane proteins are gener­ally loosely attached to the membrane and are more readily removed than are the integral proteins. Peripheral proteins are rich in amino acids with hydrophilic side chains that permit interaction with the surrounding water and with the polar surface of the lipid bilayer. Peripheral proteins on the cell’s exterior membrane surface often contain chains of sugars (i.e., they are glycoproteins).

2. Integral (Intrinsic) Proteins:

Integral or intrinsic membrane proteins contain both hydrophilic and hydrophobic regions. The hydrophilic portions of the protein interact with the polar heads of the lipid molecules at each surface of the bimolecular leaflet.

Portions of integral proteins that project be­yond the surface of the lipid bilayer are also rich in hy­drophilic amino acids. Amino acids in that part of the protein projecting from the outer membrane surface may be linked to chains of sugars. Parts of the protein that are buried in the hydrophobic portion of the lipid bilayer are rich in amino acids with hydrophobic side chains.

These side chains are believed to form hydro­phobic bonds with the hydrocarbon tails of the mem­brane phospholipids. It is speculated that within the hydrophobic interior of the membrane, the secondary structure of integral proteins is alpha helix and/or beta sheet (Fig. 15-12).

In the alpha helix conforma­tion, amino and carboxyl groups along a stretch of the polypeptide’s backbone form hydrogen bonds with one another in the beta sheet, hydrogen bonds are formed between amino and carboxyl groups in stretches of polypeptide that lie parallel to one another.

In the absence of such hydrogen bonding, these amino and carboxyl groups would have polar proper­ties, their hydrophilic nature being incompatible with the membrane’s hydrophobic interior. Alpha amino and carboxyl groups that are not “neutralized” by hy­drogen bonding would be expected only in those parts of the integral protein that extend into the aqueous milieu on either side of the membrane.

Integral Proteins That Span the Membrane:

M. Bretscher first demonstrated the existence of inte­gral proteins that span the entire membrane. In a se­ries of elegant experiments, Bretscher showed that radioactive ligands specific for membrane proteins of the erythrocyte were bound in smaller quantities to intact cells than to disrupted cells. Disruption of the cells was shown to expose portions of the membrane proteins previously facing the cell interior, thereby al­lowing additional radioactive ligand to associate with the protein.

T. L. Steck developed a technique for converting fragments of disrupted erythrocyte membranes into small vesicles that were either “right-side-out” (i.e., the external face of the membrane also formed the ex­ternal face of the vesicle) or “inside-out” (Fig. 15-13).

When proteolytic enzymes were added to separate suspensions of each type of vesicle, certain of their membrane proteins were found to be equally suscepti­ble to digestion and could therefore be enzymatically attacked from either membrane surface. These pro­teins clearly spanned the membrane. Other proteins were susceptible to enzymatic digestion only when present in right-side-out or inside-out vesicles, indicat­ing their differential distribution in the membrane’s outer and inner surfaces.

Integral proteins that span the entire membrane contain two outer regions that are hydrophilic (i.e., one at each surface of the membrane) the central re­gion is hydrophobic (Fig. 15-12). Carbohydrate asso­ciated with the hydrophilic region facing the cell’s sur­roundings is believed to play a role in maintaining the orientation of the protein within the membrane. The hydrophilic sugars, together with the hydrophilic side chains of amino acids in the out: region of the pro­tein, effectively prevent reorientation of the protein in the direction of the hydrocarbon core of the lipid bi­layer.

3. Asymmetric Distribution of Membrane Proteins:

The outer and inner regions of the plasma membrane do not contain either the same types or equal amounts of the various peripheral and integral proteins. For ex­ample, the outer half of the erythrocyte membrane contains far less protein than does the inner, half.

In addition, various membrane proteins may be present in significantly different quantities the membranes of some cells contain a hundred times as many molecules of one protein species as another. Moreover, regard­less of absolute quantity, all copies of a given mem­brane protein species have exactly the same orienta­tion in the membrane.

The differential distribution of proteins in the various regions of the plasma mem­brane within a single cell was described earlier in con­nection with liver parenchymal cells and intestinal epithelium. This irregular distribution of membrane proteins is known as membrane asymmetry. Not only are the proteins of plasma membranes asymmetri­cally distributed but so too are the proteins of the mem­branes of the endoplasmic reticulum and vesicular or­ganelles (e.g., mitochondria).

4. Mobility of Membrane Proteins:

When cells are grown in culture, there is an occasional fusion of one cell with another to form a larger cell. The frequency of cell fusion can be greatly increased by adding Sendai virus to the cell culture. In the pres­ence of this virus, even different strains of cells can be induced to fuse, producing hybrid cells or heterokaryons. D. Frye and M. Edidin utilized this phenom­enon to demonstrate that membrane proteins may not maintain fixed positions in the membrane but may move about laterally through the bilayer.

Frye and Edidin induced the fusion of human and mouse cells to form heterokaryons and, using fluorescent antibody labels, followed the distribution of human and mouse membrane proteins in the heterokaryon during the time interval that followed fusion.

At the onset of fu­sion, human and mouse membrane proteins were re­spectively restricted to their “halves” of the hybrid cell, but in less than an hour both protein types be­came uniformly distributed through the membrane (Fig. 15-14). The distribution of the membrane pro­teins was not dependent on the availability of ATP and was not prevented by metabolic inhibitors, indicating that lateral movement of proteins in the membrane oc­curred by diffusion.

Although some membrane proteins are capable of lateral diffusion, many are not. G. Nicolson and others have obtained evidence suggesting that many integral proteins are restrained within the membrane by a pro­tein network lying just under the membrane’s inner surface (Fig. 15-9). In many cells, this network is as­sociated with a system of cytoplasmic filaments and microtubules that radiate through the cytosol forming a cytoskeleton.

5. Enzymatic Properties of Membrane Proteins:

Membrane proteins have been shown to possess enzy­matic activity. Table 15-1 lists some of the enzymes that are now recognized as constituents of the plasma membrane of various cells. To this list of proteins must be added receptor proteins (such as the insulin- binding sites of the liver plasma membrane) and struc­tural or non-enzymatic proteins.

Ectoenzymes and Endoenzymes:

Enzymes disposed in the plasma membrane may be characterized accord­ing to the membrane face containing the enzymatic activity. Accordingly, ectoenzymes are those enzymes whose catalytic activity is associated with the exterior surface of the plasma membrane the activity of plasma membrane endoenzymes is associated with the interior of the cell. Many (perhaps all) plasma membrane ectoenzymes are glycoproteins.

6. Isolation and Characterization of Membrane Proteins:

Because of the relative ease with which they may be purified, the plasma membranes of erythrocytes pro­vided much of the early information on the chemistry of proteins (and lipids) present in membranes. Now, however, plasma membranes can be obtained from many cell types in a reasonably uncontaminated state using various forms of density gradient centrifugation.

Nonetheless, the individual protein constituents of the membrane are not so easily extricated for indi­vidual study because of their high degree of insolubil­ity. Varying degrees of success in extracting proteins from the plasma membrane have been achieved using organic detergents (especially sodium dodecyl sulfate, SDS) and concentrated solutions of urea, n-butanol, and ethylene diamine tetraacetic acid (EDTA).

These chemicals have a disaggregating effect on membranes and cause the release of many of the membrane pro­teins by dissociating the bonds that link the proteins together or to other membrane constituents. Often, the removal of these agents from a preparation of sol- ubilized membrane proteins is quickly followed by the reassociation or reaggregation of the proteins to form an intractable matrix.

Once solubilized, the membrane proteins can be sep­arated into discrete classes using electrophoresis, chromatography, or other procedures. This generally demands that the dissociating agents be present in the separating medium (e.g., the electrophoresis gel, the column eluent, etc.) other­wise, application of the membrane extract to the me­dium is followed by membrane protein reaggregation into insoluble complexes that will not separate into distinct fractions.

For example, the separation of liver plasma membrane proteins is achieved only if the electrophoresis gel contains SDS. The solubility problem has been one of the greatest barriers to progress in isolating and fully characterizing the proteins of membranes.

Some of the plasma membrane enzymes listed in Table 15-1 have not actually been isolated from the membrane, as removal and isolation of the enzyme is not a prerequisite for establishing its presence. In­stead, the enzyme activity can be measured directly in the (un-solubilized) membrane preparation.