Cytosolic β-Glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain

Heat linearizes the protein, and the reducing agents break the disulfide bonds that maintain the protein structure in native form. To ensure that the proteins will be separated by their molecular weights and not their charge, SDS will be used to coat the protein and give it a negative charge. This ensures that the protein is separated just by their molecular weights. It is important to note that if catalytic protein (enzymes) are being separated, they should not be treated with reducing agents or SDS. Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis or SDS-PAGE, forms a protein complex by binding to the polypeptides in the proteins. This SDS-protein complex is proportional to the proteins molecular weight. Biochemist can use this information to identify key proteins in a biochemical system. Dr. Sudakshina Ghosh uses this technique, for example, to “identify specific oxidized proteins that provide mechanistic information regarding oxidative stress” (1

For the peptide Ala-Arg-Lys-Ala-Asn-Ser-Ala-Ser, what would be the expected charges at pH 1, 7, and 13?

The GLAT enzyme itself belongs to the histidine triad super family and is a member of branch III. This enzyme shows specific nucleoside monophosphate activity and is a homodimer with each monomer containing a single domain comprised of 6 α- helices and a β-sheet which is formed by 13 antiparallel and 1 parallel strand. The mechanism of this enzyme is described as having ping-pong kinetics with the following two steps. In the first step the active site histidine attacks R-phosphorus of UDP-glucose which displaces glucose-1-phosphate and forms a covalent intermediate. The second step involves the previously formed intermediate reacting with galactose-1-phosphate to displace the histidine and produce UDP-galactose. (Facchiano, 103-104).

Corporation, W. B. (2013). Introduction to Enzymes. Retrieved 2013 йил 27-2 from Worthington Biochemical Corporation: https://gateway.emmanuel.qld.edu.au/cvpn/aHR0cDovL3d3dy53b3J0aGluZ3Rvbi1iaW9jaGVtLmNvbQ/introbiochem/effectsph.html

Price, N. C., & Stevens, L. (1999). The Cell and Molecular Biology of Catalytic Protein: Fundamentals of enzymology (Third ed.).

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

Gene BglA was sub-cloned in pET21a (+), recombinant vector transformed and propagated in E. coli BL21 CodonPlus under the control of T7/lac promoter system. The positive bacterial transformants were grown in LB-ampicillin broth (100 µg mL−1) and induced with IPTG for the production of recombinant BglA enzyme. Optimal heterologous expression and efficient folding of BglA protein was observed with 0.5 mM IPTG, after 72 h incubation at 22°C. The use of reduced concentration of IPTG for getting maximum gene expression, was proved an efficient method for the production of active soluble enzyme. The protein was purified by applying two subsequent purification steps employing ion exchange and hydrophobic interaction chromatography after heat-treatment

Two Polymerase Chain Reactions (PCR) were set up, one was digested with the restriction enzyme and the other served as the control. 11.0 µl of the forward primer solution, 11.0 µl of the reverse primer solution, and 3.0 µl of the

The second pathway is not completely independent from the first,as in this, the Amadori product (fructosamine) reacts with oxidative agents, like hydroxyl radicals, and undergoes modifications that lead to the formation of carboxymethyllysine (CML), one of the main AGEs (Ahmed et al., 1986, Booth et al., 1997, Thorpe and Baynes, 2003). The hydroxyl radicals can be produced either by transition metal-catalysed oxidation or by glucose autoxidation (Hunt and Wolff, 1991). This close interaction between glycation and oxidation leads to the development of the term glycoxidation, which describes the fact that AGEs are produced mostly from the interaction of these two processes (Baynes, 1991). Although indications and potential mechanisms are in place to suggest an active involvement of oxidative stress in protein glycation in normoglycaemia and hence the increase in the risk of chronic diseses, so far little evidence is available to support such a hypothesis.

Cancer glutamine metabolism has been a whole lot complicated to figure it out since there is flexibility of the metabolic systems as well as the number of free nutrients to which the cells have access to simultaneously. The cancer cells in culture are avid consumers of glutamine and metabolize glutamine at higher rates compared to other amino acids. Glucose on the other hand is said to play a crucial role in cancer cell metabolism and glutamine is second and both are needed for proliferating cancer cells for bioenergetics and synthesis of intermediates for macromolecular. The Warbug’s effect states that most cancer cells rely aerobic glycolysis to generate energy for cellular processes which yields less adenosine 5’-triphosphate (ATP). In

Succinyl CoA inhibits α- ketoglutarate dehydrogenase and citrate synthase. The enzyme, α- ketoglutarate dehydrogenase, catalyzes the oxidative decarboxylation of α- ketoglutarate (where equilibrium is attained far to the right towards succinyl CoA) and is allosterically inhibited by succinyl CoA. Citrate synthase is also inhibited by succinyl CoA where in the TCA cycle it is converted to oxaloacetate. Therefore, elevated generation of succinyl CoA does not just produce additional oxaloacetate; it also reduces the rate at which it is converted to citrate via inhibition of citrate synthase.

The free hydroxyl group of galactose at 6th carbon is essential for galectin binding49.Another very significant structural feature of ST6Gal-I is that it contains six cysteine residues which are also involving disulfide linkages, Cys142-Cys406, Cys184-Cys335, and Cys353-Cys364. These disulfide bonds give important features such as hydrophobicity and rigidity. The other major structural feature of this enzyme is containing a large glycan (with ten monosaccharide units) covalently bonded to Asn149. This specific glycan is also the acceptor for the reaction catalyze by ST6Gal-I. Therefore, The ST6Gal-I is acting as an enzyme as well as a

Enzymes of classes A, C and D have a serine residue at their active site that acts as a nucleophile to catalyze hydrolysis, whereas those of class B require a zinc co-factor for activity. They also share the common function of hydrolyzing the lactam ring in beta lactams, rendering them inert.

Glycosylation patterns in plants differ from those in humans and could affect the functionality of the

Glucoamylase is a hydrolysing enzyme. It can degrade both amylose and amylopectin by hydrolysing both α-1, 4 and α-1, 6 glycosidic links of starch and produce glucose. [9][10] Glucoamylase [(1, 4)-α-D-glucan glucohydrolase, amyloglucosidase, EC 3.2.1.3 is an exo-acting enzyme. It yields β-D-glucose from the non-reducing chain-ends of amylose, amylopectin and glycogen by hydrolysing α-1, 4 linkages in a consecutive manner. [11] The α-1, 6 glycosidic bonds are also hydrolysed, but at a much reduced rate. Hence,