This combination of predictable behavior and invariant fundamental attributes is what makes the physical sciences so valuable in contributing to technological advance — the electron, the photon, the chemical reaction, the crystalline structure, when confined to the controlled environment of the laboratory or the engineered design of a technology, behaves as it is supposed to behave pretty much all the time.
Calculate the WORK done by this frictional Force and include this Energy lost to friction in your analysis of the total energy of the system.
Important points that should be discussed by each member:
DA: Do not provide the raw data for part 1 of this lab.
Two major types of evidence have provided our present concept of the compartmentation of
intracellular function: (1) inferences from microscopic observation, and (2) evidence from actual
physical separation and biochemical analysis of intracellular constituents. This experiment will
provide evidence for the localization of cellular respiration in mitochondria, and that of glycolysis
in the soluble portion of the cytoplasm. The purpose of the experiment is not merely to verify that
a given function resides in a particular cell part, but rather to introduce you to an extraordinarily
important method of studying living matter and to lead you into some of the mental processes
required to infer how things work at the subcellular level.
Note that in this experiment we will be concerned with the separation of cell organelles.
Our separation will not be complete; that is to say, we will not obtain pure mitochondria, but will
exploit a rapid procedure which lends itself to easy execution while illustrating more complex
procedures used in the research laboratory. Our procedure yields a rich harvest of mitochondria
which are functional as judged by the most sensitive biochemical criterion available: the ability to
carry out ATP synthesis coupled to electron transport.
We will actually use two methods: homogenization and centrifugation. You are already
familiar with homogenization from the experiment on the enzyme action. Remember that
homogenization produces a solution of soluble cell constituents and a suspension of insoluble
constituents. The latter include intracellular organelles such as mitochondria. These may be
separated from the other organelles and soluble material by means of centrifugation. In a
centrifuge, material is spun on an axis of rotation and thereby subjected to a force (centrifugal
force) directed outward from the rotational axis. This force causes the suspended cell organelles to
move away from the axis of rotation down the length of the centrifuge tube. If the centrifuge runs
fast and long enough, the organelles will eventually become sedimented at the bottom of the tube
The rate of sedimentation varies for different kinds of suspended particles. At a given speed
of rotation, heavier and larger particles move faster than lighter, smaller ones. With elaborate
centrifugation procedures, nuclei, which are relatively large and dense, may be separated from
“microsomes” (bits and pieces of endoplasmic reticulum with any attached ribosomes).
Our choice of experimental material derives from the need for high glycolytic and
respiratory metabolism and the easiest possible separability of mitochondria from the soluble
fraction. We therefore selected insect flight muscle, specifically that of flesh flies (Sarcophaga).
As in many other flies, these have extraordinary contractile performance, causing wingbeat
frequencies of many hundreds to over a thousand cycles per second. This is energized by
glycolysis and respiration with glucose as a fuel.
Biology 155 Laboratory Supplement: 23
The most striking features of flight muscle are (1) the enlarged size of the muscle fibrils
whose contractions are responsible for the performance mentioned and (2) the equally striking giant
mitochondria. These features lend flight muscle to our present purposes for two reasons: (1) the
activity of glycolytic and respiratory enzymes is very high, permitting easy detection; and (2)
owing to the abundance and large size of the mitochondria, they can be isolated in substantial
quantity at relatively low centrifugal forces.
Baker & Allen: The Study of Biology, 3rd ed., pp. 195-232.
Keeton: Biological Science, 3rd ed., pp. 137-138, 163-180.
Loewy & Siekevitz: Cell structure and Function, 2nd ed., pp.310-314.
Raven & Johnson: Biology 4th ed., pp. 193-205.
Biology 155 Laboratory Supplement: 24
Results 20 points
Result should thoroughly summarize you data. There should be text that should describe your data that you obtained and you interpretation of that data. Include tables or figures. Tables should have descriptive titles and all columns should be labeled. Figures should have descriptive captions. Graphs must have all axes labeled. Any simple direct conclusion you can make about your data can be stated in the results. Save broad conclusion or those that require logical development for your discussion.
Special Preparations Before Coming To Lab
Carefully study the logic of the experiment (see table) in order to understand why each tube
is necessary and why each component is used. Know the function of the substrates glucose and
succinate in cellular metabolism.
Assay for Glycolysis and Respiration
To understand the assay method, it is crucial to know the meaning of the terms
glycolysis and respiration. Glycolysis is best defined as the conversion of glucose into two
molecules of pyruvic acid (pyruvate). This conversion does not consume oxygen, and
accomplishes only a partial chemical degradation of a glucose molecule, thus affording the cell
only a partial utilization of glucose’s potential as a metabolic fuel. In contrast to glycolysis,
cell respiration consumes oxygen, and pyruvic acid is completely oxidized to carbon dioxide
and water. As you should expect, both glycolysis and cell respiration are accomplished
enzymatically; the former is carried out by the glycolytic enzyme system, and the latter by the
enzymes of the Krebs cycle and electron-transport chain. NOTE: The usage of the term
respiration in some texts embraces both glycolysis and the conversion of pyruvate to carbon
dioxide and water, which requires water. The usage here conforms to that of the scientists
who established this field of biochemistry and is more convenient for a variety of reasons.
As a consequence of the points stated in the preceding paragraph, a homogenate which
is glycolyzing but not respiring will not use oxygen. If oxygen is utilized, its concentration in
solution will fall. We will use the dye, Methylene Blue to indicate when the oxygen is used
up as a result of respiration. When the concentration of dissolved oxygen is very low, the dye
becomes colorless. This happens as a result of chemical reduction of the dye. Reducing
equivalents from metabolic intermediates in the Krebs cycle are passed to NAD (or in the case
of succinate, FAD) and from these nucleotides on via the electron-transport chain. The dye
intercepts this passage and gets reduced. The dye color disappears when it is reduced.
Therefore, if we supply glucose to a homogenate in the presence of Methylene Blue
and the dye becomes colorless after a period of time, we have evidence for both glycolysis and
respiration. If instead of glucose we supply a substrate in the Krebs cycle derived from
pyruvate (we will use succinate for this purpose) and the dye bleaches, we have evidence for
respiration but not glycolysis. If we supply glucose to a homogenate capable only of
glycolysis, the dye would not be reduced and any inference as to the occurrence of glycolysis
would have to be based on other data. Be sure you understand the reasoning behind all these
The strategy of the experiment is designed to reveal which part of the muscle
homogenate carries out glycolysis and which part carries out respiration. The procedure
breaks down into four sections: (1) preparation of the homogenate; (2) fractionation of the
homogenate by centrifugation; (3) biochemical analysis of the enzymatic capabilities of the
fractions obtained; and (4) microscope observation of the fractions (optional). Students should
work in pairs. Because the preparation of the homogenate is a relatively complicated
procedure, a flow sheet has been prepared for you (see Figure 1). Read the instructions very
Biology 155 Laboratory Supplement: 25
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