* Slide 1 * The emerging method I chose to focus on was the identification of O-GlcNac modifications through the utilization of a newly constructed chemoenzymatic approach developed by the Hsieh-Wilson Lab at Cal Tech. * Slide 2 * The O-GlcNac modification is a posttranslational modification analogous to phosphorylation * OGT and OGA are the two enzymes that catalyse the reversible addition and removal of O-GlcNAc on proteins [As shown in this figure) * Numerous publications have implicated O-GlcNAc modifications as regulators with respect to enzyme activity, DNA binding, phosphorylation binding and many other biochemical processes. * Slide 3 * Prior to the construction of the development chemoenzymatic approach by the Hsieh-Wilson lab, the common method utilized for glycosylated protein labeling included the utilization of antibodies, which lacks with respect to binging affinity and selectivity. * Another method commonly utilized to identify O-GlcNAc modifications has been the utilization of radioactive labeling * In which a galactose ring with a labeling tritium is transferred to the GlcNAc group with the help of the GalT enzyme * The flaws associated with this method are price, utilization of radioactive material, and long exposure time. * The Hsieh-Wilson lab choose to adapt this radioactive labeling through utilizing GalT to transfer a galactose ring that contained chemically functionally and eliminate the use of radioactive regents. * Slide 4 * So they constructed UDP analogue 1 that posses a galactose ring with a functional ketone moiety. * The transfer of this galactose ring onto GlcNAc will be done with Y289L GalT, a GalT mutant that has been shown to posses a larger binding pocket that increases catalytic activity with respect to larger substrates. * The ketone group on the galactose group can subsequently undergo oxime ligation with the amine terminal of amooxy biotion label. * Slide 5 * Oxime ligation is selective with respect to other macromolecules in the cell. * Focusing on the ketone on the UDP 1 * First there is a nucleophilic addition * Followed by protanantion and subsequent dehydration of the addition product. * Which can then be probed with streptivin for chemiluminescence visualization and protein isolation. * Slide 6 * So once the group strategized their chemoenzymatic approach they employed it on a known glycosylated CREB peptide. * LC-MS was utilized to monitor the progression of the reaction * Initially we observe glycosylated peptide, confirmed via mass * After incubation of CREB peptide with GalT mutant, and UDP analogue 1 we observe complete keto-sugar transfer. * A subsequent biotin labeling can be observed in the lower chromatogram * Slide 7 * After demonstrating the ability to label a single peptide, the group wanted to demonstrate the selectivity of their method with respect to the entire CREB protein, known to be highly glycosylated. * Point out band observed in lane one when all reagents were utilized. * And lane 4 utilized glycosylated CREB, demonstrating this method is specific to GlcNAc modifications. * Slide 8 * As a result of demonstrating the selectivity of their labeling method utilizing CREB protein, the group wanted to test the sensitivity of their method utilization alpha crystillan protein * Prior publications have…
Enzymes are the catalysts of biological systems. They speed up chemical reactions in biological systems by lowering the activation energy, the energy needed for molecules to begin reacting with each other. Enzymes do this by forming an enzyme-substrate complex that reduces energy required for the specific reaction to occur. Enzymes have specific shapes and structures that determine their functions. The enzyme’s active site is very selective, allowing only certain substances…
What Are Enzymes?
Substances that speed up chemical reactions are called catalysts. Organic catalysts are called enzymes.
Enzymes are specific for one particular reaction or group of related reactions.
Many reactions cannot occur without the correct enzyme present.
They are often named by adding "ase" to the name of the substrate. Example: Dehydrogenases are enzymes that remove hydrogen.
An enzyme-substrate complex forms when the enzyme’s active site binds with…
piece of information that can be verified such as whether the bread tastes good: false
17. It is always easy to determine if an entity is living. False
18. Organs of the body having similar function are called what? System
19. What is one cause of genetic variation of parents which can be inherited by the offspring? Mutations
20. Connective tissue is considered a tissue because it carries signals: false
21. A group of similar organisms of the same species is called what? Population
22. A theory is…
GENETICS I: GENE ACTION, GENE REGULATION, AND MUTATION
1. A typical prokaryotic cell contains __________chromosome(s).
2. The basic building block of a DNA molecule is the__________________________
3. The observable physical traits of an organism are collectively referred to as its ______________________
4. An organism's genetic makeup is its_____________________________
5. The nitrogenous bases found in DNA are ____________, ______________,_____________and ______________.…
Alleles are pairs of genes found in certain positions in the chromosomes (see figure 1). Chromosomes are threadlike strands of DNA and protein. Chromosomes are located in the nucleus of most living cells carrying genetic information in the form of genes (see figure 2). Alleles determine the genotype of an organism. The Genotype shows what alleles an organism has by letters either being upper or lower case (see figure 3). These DNA codings determine different traits that the parents…
Enzymes, their activity and their role in the body.
P5 - Demonstrate the factors that affect enzyme activity.
Enzymes are biological catalysts. This means that they are substances with the ability of increasing the rate of chemical reactions without being used up themselves. They are also proteins that are folded into complex shapes (tertiary proteins) allowing smaller molecules to fit into them. The molecules that react with the Enzymes are called “Substrate molecules”. The substrate molecules…
Proteins are a class of organic compounds which are present in and vital to every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together, protect, and provide structure to the body of a multi-celled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and various lipoproteins, they effect the transport of oxygen and other…
is always lost)
Structure and function are interrelated – or – form follows function
Maximize surface area for absorption or energy production.
– an organisms basic unit of structure and function
–enclosed by a membrane
– use DNA as genetic information
– lowest level that can preform all activities required for life.
Two Types of Cells
Eukaryotic – has nucleus and organelles (all other life forms)
Prokaryotic – doesn’t have nucleus or organelles (Achaea & Bacteria). Most are single…
epithelial cells is sticky because it has less water than normal, this is because of less salt & water transport caused by a faulty protein channel.
Antibodies, enzymes and many hormones are all protein molecules. Proteins also have functions within a membrane.
Proteins are composed of amino acids, there are 20 different amino acids that occur in proteins.
All amino acids contain an amine group -NH, a carboxylic acid group -COOH, and a hydrogen -H attached to central carbon atom…