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BCH 3120 Lecture Notes - Pyruvate Dehydrogenase Complex, Glycerol Kinase, Pyruvate Dehydrogenase

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School
University of Ottawa
Department
Biochemistry
Course
BCH 3120
Professor
Elaine Beaulieu

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Document Summary

If we were to be at equilibrium, we would be a gas ball, cause we would be at equilibrium with air which wouldn"t work bc all of our biomolecules would dissipate. Our body works to keep its complexity so we are in a dynamic steady state. your body will not start making bits that they don"t need unless its energy that will be stored. Proteins have half-lives, in different tissues, different proteins have different functions. 0. 9 days so the proteins are being laid down quickly. So virtually, your whole body is being re made. In the universe there is always the same amount of energy, it can only be transformed. Nutrients in the environment are what heterotrophs use in order to create complex molecules. So we transform complex molecules into simple molecules. We also have a little bit of heat, we need this to maintain our body temperature.

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Related Questions

question 1

What would be the best guideline to a healthy diet according to your instructor which is validated by other resources.

1.

Eat organic foods and primarily non-GMO foods.
2.

Eat all your meals at home.
3.

Eat a variety of foods.
4.

Eat a vegetarian diet.

5.00000 points

QUESTION 2

A complete protein has all the 20 amino acids: 9 are essential for our body to consume and 11 our body makes on its own. We need 9 + 11 = complete protein to build muscle, repair cells, keep our immune system working properly, etc.

True

False

5.00000 points

QUESTION 3

It shouldn't be hard to reverse the GMO guidelines in this county.

True

False

5.00000 points

QUESTION 4

Out of a variety of foods you could eat, which one will make you feel the most full?

Milkshake

Salad

Hamburg

Beer, wine or hard liquors

5.00000 points

QUESTION 5

Which food has been identified as the food that cultures and people who have lived a very long life have always had in their diet?

Chocolate

Vegetables

Whole grains

Beans

5.00000 points

QUESTION 6

When we eat animal products: fish, turkey, chicken, duck, pork, beef, eggs, etc., these proteins when cooked give the body all the amino acids it needs to do the bodily functions that are dependent on protein.

True

False

5.00000 points

QUESTION 7

Trying new foods has value. Different foods gives the body more nutrition than having the same foods everyday. Your perception of food does change, thus how many times do you recommend to your family and friends to try a food they may not normally like throughout their life?

2-3

90-100

7-8

20

5.00000 points

QUESTION 8

Supplements should be taken with caution because they are unregulated by the FDA, they do not fall in the category of being food or medication.

True

False

5.00000 points

QUESTION 9

There is little scientific evidence that organic foods are more nutritious than conventionally grown foods.

True

False

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacIQ, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

-------------------------------------------------------------------------------------------------------------------

DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg2+ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

-------------------------------------------------------------------------------------------------------------------

DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

-------------------------------------------------------------------------------------------------------------------

DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

-------------------------------------------------------------------------------------------------------------------

DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

Human body can be described by many means – one of them – it is relatively big vessel with complex body fluid. For diagnostic purpose it is very complicated to detect insignificant number of biomarkers on very early stages of pathological process. This article, present pioneering method to initiate and conduct diagnostic by researching body fluids.

**With additional research provide additional examples of scientific way of dealing with solutions to improve functionality of human life. (QUESTION - This is exactly what is witten on my assignment)**

Analyzing human blood for a very low virus concentration or a sample of water for a bioterrorism agent has always been a time-consuming and difficult process. Researchers at the Georgia Institute of Technology and Emory University have developed an easier and faster method to detect these types of target molecules in liquid samples using highly porous, micron-sized, silica beads. The researchers developed a technique to simultaneously or sequentially add optical and magnetic nanoparticles into the beads. Adding magnetic nanoparticles allows the use of a magnetic field to attract and easily remove the beads from a liquid sample. “These nanoparticles enter the pores of the microbeads so quickly and so completely -- essentially more than 99 percent of the nanoparticles go into the pores of the beads,” explained Shuming Nie, the head researcher on the project and the Wallace H. Coulter Distinguished Chair in Biomedical Engineering and director of Emory-Georgia Tech Nanotechnology Center. The beads are mixed in a liquid such as urine. Viruses, proteins or other biomarkers are captured on the bead surface. After the beads are removed from the liquid, optical imaging is used to determine the concentration of a specific protein or virus in the liquid sample based on the number of proteins or viruses attached to the surface of the beads. Tushar Sathe, a graduate student in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, described the process of creating these novel beads and their clinical applications on Jan. 20 at SPIE Photonics West in San Jose, California. The work was also published in the Aug. 15 issue of Analytical Chemistry. The technology involves embedding fluorescent quantum dots and magnetic iron oxide nanoparticles inside the beads to create dual-modality magneto-optical beads. Nie and Sathe synthesize the quantum dots in different colors by varying their size, giving the beads a unique optical signature. Having different color beads allows the researchers to detect several target molecules at the same time in the same liquid sample. “We use the quantum dots to create a set of beads that are unique and can be distinguished from each other. It’s similar to bar-coding -- once you barcode the beads and put them in the urine or blood sample, you can remove them and decode what proteins or viruses have attached to individual beads based on their spectral signature,” explained Sathe. The process of creating these beads is quite simple, according to Sathe. The surface of the beads contains a long-chain carbon molecule that makes the beads hydrophobic, meaning they repel water. The beads are dissolved in butanol and washed several times. Then the beads are counted and optical and magnetic nanocrystals are added to the suspension either simultaneously or sequentially. After 15-20 minutes, the butanol is removed to get rid of any remaining nanoparticles that didn’t get incorporated into the beads and the beads are washed with ethanol. Then the beads are coated with a polymer that creates a hydrophilic surface on the beads. This allows the beads to be functionalized by adding antibodies or DNA molecules to the surface that will capture the target molecules. These beads are dual-function -- both optical and magnetic -- but according to Sathe, more functions can be added to the beads. “Adding them is as easy as adding the nanoparticles into the solution. You just have to make sure the nanoparticle surface is hydrophobic so that it interacts with the beads,” said Sathe. The primary biomedical applications for this new technology will be to detect cancer and neurological diseases by identifying certain molecules present in human blood or urine that indicate specific diseases, according to Nie, who is also professor of biomedical engineering, chemistry, materials science & engineering, and hematology and oncology at Emory University and the Georgia Institute of Technology. “Some of the biomarkers for Alzheimer’s disease have very low concentrations in the blood so you need highly sensitive techniques that can find a specific molecule to diagnose this disease,” explained Nie. “Our technique could also be used to monitor therapeutic response. For example, if the viral level decreases in samples taken at later dates, then we know the drug is probably working.” This new technology allows the researchers to analyze very low concentrations of target molecules. “Instead of analyzing a liter of sample where the concentration could be very dilute and you might not see the target molecule you’re looking for, you can let the beads capture the molecules on their surface, remove them from the liquid, and then just measure the number of molecules attached to the beads,” said Nie. This ongoing research is funded by the National Cancer Institute, the Department of Energy’s Genomes to Life (GTL) Program, the Department of Defense and the Georgia Cancer Coalition, a public-private partnership established by the Georgia General Assembly in 2001.