Biology Reference
In-Depth Information
peculiarities, countless procedures have been published. This chapter will consider a few of
the problems encountered in isolating membranes and some of the more common techniques
that have been employed.
As discussed in Chapter 1, a eukaryotic cell is composed of an astonishing number of
different membranes, all packed tightly into a very small volume. Membranes present
a tiny and ever changing target for investigation, making their isolation particularly chal-
lenging. Every eukaryotic cell is surrounded by a relatively tough plasma membrane (PM)
that encumbers countless more delicate internal membranes. This presents a dilemma.
How does one break open the PM without severely impacting the tightly packed, intercon-
nected and delicate internal membranes? An old analogy seems appropriate. You are given
a bag of different types and sizes of watches that had been smashed with a sledgehammer
into thousands of intermingled, broken parts. From this you are asked to reassemble the
watches and determine how they measure time!
Instantly upon breaking open a cell, the internal membranes are relieved of curvature
stress by taking new morphologies, often in the form of similar-sized vesicles called micro-
somes
[1]
.
Microsomes
The concept of microsomes is critical to membrane studies. Microsomes are small sealed
vesicles that originate from fragmented cell membranes (often the endoplasmic reticulum
(ER)). These vesicles may be right-side-out, inside-out or even fused membrane chimeras.
Microsomes may have unrelated proteins sequestered in their internal aqueous volumes or
attached to their surfaces. Microsomes are therefore artifacts that arise as a result of cell homog-
enization and are very complex. By their very definition, microsomes
per se
are not present in
living cells and are physically defined by an operational procedure, usually differential centri-
fugation (discussed below,
[2]
). In a centrifugal field, large particles including unbroken cells,
nuclei, and mitochondria sediment out at low speed (
10,000g), whereas much smaller micro-
somes do not sediment out until much higher speeds (~100,000g). Soluble cellular components
like salts, sugars, and enzymes remain in solution at speeds that pellet out microsomes.
Microsomes have been observed, if not understood, for a long time. In an early review
from 1963, Siekevitz linked microsomes to remnants of the ER after cell homogenization
[3]
. Typically, discussions of microsome properties and isolation procedures are found buried
in papers whose primary objective is to investigate a specific integral membrane protein (for
example see
[4]
).
Once broken, all membrane fractions are instantly exposed to new osmotic stresses, diva-
lent metal ions, unnatural pHs, and degradative enzymes including proteases, oxidases,
lipases, phospholipases, and nucleases
[2,5
<
7]
. Historically, sucrose has been the major
osmotic component (osmoticum) of cell homogenization buffers
[8]
. Sucrose is inexpensive,
readily available in pure form, and is poorly permeable to most membranes. Importantly, it
does not destroy enzymatic activity. In many contemporary membrane isolations, Ficoll
e
(GE Healthcare companies) has replaced most or all of the sucrose. Ficoll is an uncharged,
highly branched polymer formed by the co-polymerisation of sucrose and epichlorohydrin
[9]
. Due to its multiple (
OH) groups, Ficoll, like sucrose, is highly water-soluble. Often
a little sucrose is added to the Ficoll to accurately control the density, viscosity, and osmotic
e
