Azide Lab Report

Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay.

The purpose of this lab was to identify unknown bacteria cultures using various differential tests, and my unknown bacteria is #17. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were Gram stain, Catalase, Mannitol Salt Agar (MSA), Blood Agar, Novobiocin, Coagulase, and DNAse (Alachi, 2007).

Table 2: Consists of color extract taken from a red cabbage for a natural indicator. The pH reading that was measured by using the pH meter and the result of the pH reading to determine whether the solution was acidic or basic.

Specifically, the secondary antibody Alexa 546 Chicken Anti-RatDAT was used to increase reactivity by amplifying the signal; occurring as numerous secondary antibodies bound to a single primary antibody. The increase in binding Alexa 546 Chicken Anti-RatDAT to a Rat Anti-DAT’s Fc region illustrated a direct correlation to increased steric hindrance; meaning the attachment of secondary labels to primary antibody molecules resulted in a decrease of antibodies binding to a surface antigen. In order for Rat Anti-DAT to be able to bind with DAT, Triton X-100 was used to lyse cells for the extraction of proteins or organelles, or to increase the permeability of the membranes of living

The diverse roles of amino acids: 1. Hormones (signaling type molecules)  Dopamine, thyroid type hormones 2. Metabolic Intermediates  Catabolic amino acids  Citrulline: key intermediate in the urea acid cycle, the pathway by which mammals excrete ammonia.  Ornithine: in urea acid cycle, precursor to arginine, disposal of nitrogen. 3. Osmotic effects (Osmolytes- organic compounds affecting osmosis, soluble in solution within a cell, play a role in maintaining cell volume and fluid balance.)  Ex: Antarctic fish utilize stores of amino acids to offset preservations of different ions. 4. Buffering Capacity  Zwitterion (means twin in German): A zwitterion is a chemical compound that is electrically neutral but carries formal positive and negative charges on different atoms. Zwitterions are polar and usually have a high solubility in water and a poor solubility in most organic solvents. o Often times the R group is charged.  Ex: Histidine (essential amino acid) the R group has a charge, pKa at pH 7.4. Modest change in pH results in change in charge; many proteins have a Histidine residue sitting in the active site. Changes ability to interact with substrate, which causes a profound change in kinetics.

Although the unstatutable albuterol majority of total flux is paracellular, the cellular uptake; on the other hand, is saturable and its action contributes to the net flux. If administered in large doses, it has adverse effects and this last can activate Beta 1 receptor which action causes nefast cardiac stimulation. The writers' group also studied whether b2-agonist interact with organic cation transporters (OCT) and whether this interaction exerted an influence on their passage across the respiratory epithelium to their target receptors.

From these experiments, we were able to produce a standard curve, and a Lineweaver-Burk plot to determine Km (constant) and Vmax (maximal velocity). The turnover number (Kcat) was measured for pure beta- galactosidase from an assay at 280 nm absorbance. Additional we could determine the IPTG mechanism of inhibition, determine KI/KI’, and the optimal pH for beta- galactosidase.

Acidosis influences L-glutamate carrier proteins and changes the 3-dimentional structure of the transporter which may finally halt L-glutamate transport (71). Furthermore, acidosis can change the ratio of OH-in to OH- out. This ratio plays an important role in the L-glutamate transporting system on neurons (72) and in turn its disturbances will inhibit L-glutamate (70, 73, 74). Acidosis also stimulates L-glutamate release and this stimulation may participate in the mechanisms by which acidosis inhibits L-glutamate uptake (70, 75). When the stimulation of L-glutamate release and its (re-)uptake inhibition occur simultaneously, it probably leads to an increase in extracellular glutamate which subsequently activates NMDA and AMPA receptors and cause excessive influx of Ca2+ and excitotoxicity (69, 76) Consequently, these NMDA receptors trigger more changes in pHi which seems to be toward acidification. Such a shift results from increases in intracellular sodium and extracellular potassium which is mediated by NMDA receptor activation and subsequently stimulates glycolysis in neurons (77). The mentioned pHi acidification is accompanied by the increased production of lactate whose pathological concentrations may play a role in NMDA receptor-induced neuronal injury or death and neurodegeneration (77-80).

Glucose transport proteins are found in various isoforms. GLUT1 is the most universally distributed of the glucose transporter isoform. It is highly expressed in erythrocytes, central nervous system, blood-brain barrier and placenta. Glucose transport occurs via GLUT1 in various cells that form barriers between body tissues and the blood supply. This transporter has a high affinity and a low Km of 1, meaning GLUT1 transporters in cell membranes are increased by reduced glucose levels and vice versa (Kraegen et al., 1993). Therefore, tissues with GLUT1 are in constant need of glucose supply.

This process called the “uncoupled”. “Down regulation” means the receptor mRNA and protein synthesis decrease and the preexisting receptor degradation. The “uncouple” and “down regulation” are both involved in the receptor desensitization, but in different steps. These steps are following described. First, the receptor uncouple from heterotrimeric G proteins; Second, cell surface receptors internalize into intracellular membranous compartments; Third, receptor mRNA and protein synthesis decrease result in the down regulation of the total cellular complement of receptors, as well as both the lysosomal and plasma membrane degradation of pre-existing receptors.

Zinc ions in protein interface may influence the formation of stable quaternary structures, as in the case of hexameric insulin, which is formed from three protein dimers and two Zn2+ ions. As has been said, zinc coordination in proteins varies widely, with a strong preference for histidine, cysteine, aspartate and glutamate. Despite these different coordination surroundings, proteins have high affinity towards zinc ions. (6)

The transport activity is expressed as nmoles of substrate transported during the incubation time per milligram of the reconstituted protein and it is calculated with the following formula:

These transporters are divided into two families. One of them is responsible for transporting metals to the cells after ingestion to help the cell perform its functions by binding to the specific protein there for example binding to Cu/ZnSOD protein to breaks the free radicals20,21. The other one transports the metal that binds with glutamatergic neurotransmitter from the presynaptic vesicle to the postsynaptic one and those metals are responsible for learning, cognition, and memory functions by regulating the excitation process by either inhibition of NMDA and GABA receptors or enhancing AMPA receptor in

Heterodimers play an important role in signal transduction and alterations of receptor pharmacology G-protein coupled receptors (GPCRs) are the most prominent among receptor families to heterodimerise. The example of GPCR heterodimerisation is the γ-amino butyric acid (GABA) b receptor which is composed of 2 subunits, either the GABAb1A or GABA1B isoforms along with the GABAb2. This heterodimerisation is important due to the presence of a high affinity binding site for GABA on the b1A and b1B Isoforms which is in contrast to the low affinity binding site on the GABAb2 receptor subunit. The heptahelical domain (HD) of the GABAb2 receptor transmits the signal to the heterotrimeric G-protein which interacts with the intracellular loop i2 and i3 of the GABAb2 subunit. In addition to signal transduction heterodimerisation also helps the receptor to be trafficked to the membrane surface. There is a C-C coiled interaction in the intracellular carbon tail which masks the E.R retention signal and allows the receptor to

Ceramides are an example of a family of cellular lipids (Yasuda et al., 2001). Ceramides are synthesised at the endoplasmic reticulum from precursor compounds and are transported to the Golgi apparatus for conversion into one of the several sphingolipids (Yasuda et al., 2001). The transport of such compounds is highly selective if not specific (Kumagai et al., 2005; Yasuda et al., 2001). Inhibiting transport is a useful tool in investigating the role of substrates (Yasuda et al., 2001) and makes an attractive target for biochemical manipulation of the cell (Ueno et al., 2001).