BIOL 340 Molecular Biology
Lectures 7 and 8 Jan. 31 and Feb. 2, 2001
"Introduction to Cells"
Reading: Chap. 5 Lodish et al.
Outline:
1. Cell Theory
2. Subcellular Fractionation
3. Membrane Structure
4. Cell Organelles
1. Cell Theory:
A. All organisms are composed of one or more cells. Based on microscopic
observations of cells by Schleiden and Schwann, 1839.
B. Cells are alive and are the basic living unit of function and organization
of all living things. Based on observations of dividing cells by Virchow, mid19th century.
2. Subcellular Fractionation: Use differential centrifugation to separate a cell
extract into fractions containing different organelles.
A. Prepare homogenate (mixture of broken cells and organelles) of a tissue in
isotonic sucrose.
B. Centrifuge at 600xg 10 min.
Pellet = nuclei.
C. Centrifuge supernatant at 15,000x g 5 min.
Pellet = mitochondria (contaminated with similar sized organelles such as
lysosomes and peroxisomes).
D. Centrifuge supernatant at 100,000x g 60 min. Requires ultracentrifuge.
Pellet = "Microsomes" = mixture of endoplasmic reticulum, Golgi apparatus, and
plasma membrane.
E. Centrifuge supernatant 300,000x g 2 hr in ultracentrifuge. Pellet = ribosomes.
Supernatant = cytosol.
Assess purity of subcellular fractions by electron microscopy, Western blot (or
other immunological methods), or assays for marker enzymes.
Some key marker enzymes:
Mitochondria:
Lysozymes:
Peroxisomes:
succinate dehydrogenase
acid phosphatase
catalase
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3. Membrane Structure
A. Lipid composition
--Phospholipids
--Phosphoglycerides
--Sphingomyelin (lacks glycerol)
--Cholesterol
Carbohydrates
--Glycoproteins
--Glycolipids
B. Properties of membrane
--Phospholipid bilayer: acyl groups toward interior, phosphates or other
charged groups are on the surface in contact with water.
--Many of the lipids in the plasma membrane are freely mobile.
--Proteins imbedded in membrane.
--Integral membrane proteins = transmembrane proteins, span lipid bilayer
--Extrinsic membrane proteins, included on one face or the other of
membrane.
--Extracellular side: Proteins often contain carbohydrate residues
glycoproteins.
--Proteins are generally able to move laterally in membrane unless
tethered to another protein.
4. Membrane-Bound Cell Organelles (Animal Cell)
A. Lysosomes:
--Single membrane bound organelles found in the cell cytoplasm
--Several hundred/cell
--slightly smaller than a mitochondrion
--interior contains degradative enzymes ("acid hydrolases")
--nucleases: degrade DNA and RNA
--proteases: degrade proteins
--phosphatases: remove phosphates
--interior pH around 4.8 (acidic)
--a primary lysosome has no interior particles
--a secondary lysosome is one that has fused with other membrane
organelles
B. Peroxisomes:
--single membrane bound organelles found in cell cytoplasm
--present in all animal cells except red blood cells
--contain oxidases: enzymes that use oxygen and produce hydrogen
peroxide
--contain catalases which break down hydrogen peroxide--these often
appear as a crystalline lattice in EM
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--oxidize fatty acids to release heat and produce acetyl groups used in
biosynthesis of cholesterol
C. Mitochondria:
--double membrane bound organelle
--outer membrane allows larger molecules to pass than the inner
membrane
--may make up 25% of the volume of the cytoplasm
--main site of ATP production via aerobic respiration
-Kreb's cycle occurs in the mitochondrial matrix (interior
compartment)
--electron transport chain and oxidative phosphorylation take place
in inner membrane
--contain their own DNA (mitochondrial DNA) that encodes some genes
--loss of mitochondrial integrity leads to cell death (apoptosis)
D. Endoplasmic Reticulum:
--network of interconnected membrane vesicles extending from the
nucleus toward the plasma membrane in the cytoplasm
--smooth ER lacks ribosomes, mainly present in liver for detoxification
--rough ER is studded with ribosomes, synthesize secretory proteins and
those destined for the ER, Golgi and plasma membrane
--involved in lipid synthesis (SER) and protein processing (RER)
E. Golgi Vesicles:
--diffuse organelle consisting of a series of flattened sacs in the cytoplasm
--carry out sequential modifications of secretory and plasma membrane
proteins such as the addition of lipid and carbohydrate groups
--proteins travel through the Golgi from ERcis regionmedial
regiontrans regionplasma membrane
F. Nucleus:
--largest membrane bound organelle in the cell
--bounded by two membranes
--contains the DNA in chromosomes
--site of replication, recombination, transcription, and RNA splicing
--large molecules are transported in and out of the nucleus through
nuclear pores
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