The use of homogenization techniques and differential centrifugation has made possible the study of many isolated, membrane bound organelles. The nucleus, mitochondria and various plastids were obvious targets for analysis, since they are relatively large, and are visible with the light microscope.
Smaller structures, and particularly those associated with internal membranes, were more difficult to analyze. In 1931, W.H. Lewis observed the uptake of neutral red dye into cell vacuoles, 1 and coined the term PINOCYTOSIS, literally cell drinking. Lewis was interested in the small membrane bound organelles found within plants cells, and named vacuomes by the French Botanist, P.A. Dangeard in 1919. The term vacuome described a structure or structures in plant cells which were associated with the tonoplast (the membrane surrounding a large central vacuole) and which was apparently part of an internal membrane system of communication and flow. Figure 7.1 presents an extended view of the vacuome concept, which was developed to explain both pinocytosis and its related phagocytosis, cell eating . The study of vacuomes was an early attempt to define the function of internal membrane bound vessicles as components of membrane flow.
Several years later, the concept of an organized system of internal membranes was extended to animal cells. Our perspective on membrane bound vessicles was altered significantly in 1949 when Christian DeDuve analyzed the activity of the enzyme acid phosphorylase during a study of insulin activation. 2 Acid phosphorylase is a useful indicator of enzymatic digestion in cells. DeDuve was interested in how cells digested substances which were either pinocytized or phagocytized, and specifically, what the relationship of digestion was to the small vacuoles, MICROSOMES, which could be isolated via centrifugation. As DeDuve explains, the lab purchased an expensive and new ultracentrifuge capable of isolating small particles and even molecules. DeDuve discovered that acid phosphorylase was associated with specific microsome fractions and that the activity of the enzyme could be released by the actions of blenders, hypotonic media, freezing/thawing or detergents. As work progressed, several other enzymes were localized within these microsomes. In 1955, DeDuve et al published the now classic paper describing for the first time, a new organelle, the lysosome . 3 DeDuve was later to be awarded a Nobel Prize for this pioneering work.
The lysosome is one type of microsome; its role in cell digestion brought together a story of internal membrane flow which is integrated with the twin processes of ENDOCYTOSIS and EXOCYTOSIS of the plasma membrane. The lysosome was seen as a part of an elaborate system, that could be fractionated, but which only made sense in the whole. Pinocytosis, Phagocytosis, Golgi Complex, Endoplasmic Reticulum and Plasma Membrane where all part of this system. Figure 7.2 presents our current concept of how a cell's internal membranes are organized.
The exercises in this chapter deal with the isolation and analysis of different types of microsomes. Careful homogenization of the cell is critical for the subsequent separation of microsome fractions. Lysosomes, for example, can be isolated from rat liver in either an isosmotic sucrose media (0.25 M), or in a hyperosmotic sucrose (0.88 M). With the use of isosmotic solutions, mild homogenization is required to prevent rupturing of the lysosome. Excessive membrane damage also results in the creation of high levels of contaminating vessicles formed from broken plasma membranes, endoplasmic reticulum, and nuclear envelopes.
The physical properties of isolated organelles can be determined and their function inferred from their chemical compositiion. Table 7.1 lists some of the properties of microsomes isolated in isosmotic media.