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The investigators studying the protein changes during aging examined the enzyme activity in the cells extracted from the Caenorhabditis elegans, a nematode worm. The cell extracts were then treated to conserve the enzymatic activity. Although, the investigators noted that some of the proteins were broken down by the process of extraction. The centrifugation of the extracts was performed and seven fractions were collected for isolating the location of the protease enzyme activity, the enzyme called cathepsins. The fraction and the eukaryotic cellular structure in which the enzymatic activity is most active.
Introduction:
The Caenorhabditis elegans is a transparent nematode, which is free-living and it is approximately 1mm (millimeter) in length. It resides mostly in the temperate soil environments. It is the unsegmented pseudocoelomate and also does not have respiratory systems. The males have a special tail and spicules are present on the tail for mating purpose.
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Biology: The Dynamic Science (MindTap Course List)
- Penicillin is hydrolyzed and thereby rendered inactive by penicillinase, an enzyme present in some resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6 kd. The amount of penicillin hydrolyzed in 1 minute in 10 ml solution containing 10–9 g of purified penicillinase was measured as a function of the concentration of penicillin. Assume that the concentration of penicillin does not change during the assay. [Penicillin] µM Amount hydrolyzed (nanomoles) 1 0.11 3 0.25 5 0.34 10 0.45 30 0.58 50 0.61 a) Plot V0 versus [S] and 1/V0 1/[S]. Does penicillinase appear to obey Michaelis-Menten kinetics? b) What is the value of KM? c) What is the value of Vmax? d) What is the turnover number of penicillinase under these conditions?arrow_forwardThe objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O F-P=0 O CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme -CH,—O CH(CH3)2 O <=0 O CH(CH3)2 DIP-Enzyme Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol.…arrow_forwardTo approximate the concentration of enzymes in a bacterial cell, assume that the cell contains equal concentrations of 1,000 different enzymes in solution in the cytosol and that each protein has a molecular weight of 100,000. Assume also that the bacterial cell is a cylinder (diameter 1.0 μm, height 2.0 μm), that the cytosol (specific gravity 1.20) is 20% soluble protein by weight, and that the soluble protein consists entirely of enzymes. Calculate the average molar concentration of each enzyme in this hypothetical cell.arrow_forward
- Small molecules are used as inhibitors of protein action - as drugs. They most often do this by blocking the active site within the protein. Potential drugs can be screened computationally to determine if they are strongly bound to the protein. Figure 1 shows a possible conformation of a candidate drug molecule, 4-bromo-2- carboxymethylamide-pyrrole (abbreviation: BCMAP) at the active site of a protein (abbreviation: PR). Figure 2 shows the full protein structure whilst figure 3 shows a known inhibitor of the protein at the site, overlayed with another calculated conformer of BCMAP. (a) Explain what types of interactions, both intermolecular and intramolecular, that a molecular mechanics forcefield must be able to describe in order to be able to accurately determine the geometry of BCMAP in the protein. Identify which interactions will be the most important to describe accurately. Figure 1.4-bromo-2-carboxymethylamide-pyrrole (BCMAP) (C, N, O, and Br atoms in yellow, blue, red, and…arrow_forwardTwo-dimensional gel electrophoresis of proteins in a cell extract provides a qualitative way to compare proteins with respect to intra- cellular abundance. Describe a quantitative approach to the deter- mination of number of molecules of an enzyme per cell.arrow_forwardTwo-dimensional gel electrophoresis of proteins in a cell extract provides a qualitative way to compare proteins with respect to intracellular abundance. Describe a quantitative approach to determine the number of molecules of an enzyme per cell.arrow_forward
- One of your colleagues has obtained a sample of muscle phosphorylase b that is known to be relatively inactive. She has approached you for advice on how to set up an appropriate assay. She has the following items available, not all of which are appropriate for this study. Help her out by selecting the items that she should use and what their purpose is in the assay, and then explain why each of the other items would not be useful. 1. 100 UM AMP 2. 100 UM GTP 3.100 uM glucose 4. 100 uM glucose 6-phosphate 5. Branched glycogen 6. Amylose (i.e. unbranched glycogen) 7.50 mM HEPES buffer, pH 7.5 8. 50 mM potassium phosphate buffer, pH 7.5 Write out a short explanation for each of the above items, and upload your answers by the due date.arrow_forwardIdris has successfully extracted enzymatic proteins from the fish viscera (intestines and stomach). After homogenization and centrifugation, he managed to pool the crude enzyme extract. He is characterizing the enzymes. Please help Idris by answering the followingquestions:The experiments I conducted showed that one of the enzymes was successful in degrading casein.(a). Which family of enzyme is it from?(b). What is the possible name of this enzyme and why?arrow_forwardA. Lineweaver-Burk plot of the enzyme with increasing amounts of substrate in the absence or the presence of the inhibitor is shown below. Graph A : x-intercept Graph B : x-intercept = - 0.012, y-intercept = 0.8 Graph C : x-intercept = - 0.027, y-intercept = 0.8 Graph D : x-intercept = - 0.039, y-intercept = 0.8 - 0.007, y-intercept = 0.8 Graph A 4 Graph B Graph C Graph D 1 -0,04 -0,02 0,00 0,02 0,04 1/[Substrate] (uM) (i) Which graph indicates an enzymatic reaction without inhibitor? (ii) Which type of inhibitor is it? Briefly explain. (iii) Which graph indicates the highest concentration of inhibitor? (iv) Calculate the Vmax and Km of the graph showing an enzymatic reaction with the lowest concentration of inhibitor. Show the steps of calculation and unit in your answers. Keep 2 decimal places in your answers. 1/Rate (umol/min)arrow_forward
- Match the enzymatic activity with the proper description within the ubiquitin-proteasome pathway. Drag and drop options on the right-hand side and submit. For keyboard navigation... SHOW MORE ✓ E1 E2 E3 Proteasome = ||| = ||| = ||| Degradation Ubiquitin-activating enzymes Ubiquitin-conjugating enzymes. Ubiquitin ligasesarrow_forwardA newly developed drug is suspected to act by inhibiting its enzymatic target. To test this, the rates of reaction in the presence and absence of the new drug were determined. The results of these experiments have been plotted as a double reciprocal plot shown in figure 1 below. Using this figure, calculate the values of Vmax and Km for the enzyme in the presence and absence of the drug. Based on the figure and your answer, what type of inhibition is the new drug displaying? Briefly explain how this type of inhibition exerts its action on an enzyme.arrow_forwardTo answer this question, please reference the Problem Solving Video: pH and pK, of an Amino Acid. Proteases are enzymes that cleave peptide bonds using general acid-base catalysis. General acid-base catalysis relies on a protor donor or acceptor other than water. Proteases rely on proton transfer to create strong nucleophiles from active-site amino acid residues. In the protease chymotrypsin, an active-site serine is a potent nucleophile. A nearby residue, His 57, interacts with serine to increase its reactivity. A schematic of chymotrypsin's active site illustrates the active site Ser 195 and His 57 R groups. The Asp 102 residue helps position the His 57 residue via hydrogen bonding. Asp 102 [His] [His+] His 57 6.0 -H-N Chymotrypsin is a digestive enzyme with a catalytic optimum between pH 7.8 and 8.0. However, due to the presence of gastric juice, chymotrypsin often functions in an environment of pH 5.5-7.0. Incorrect The imidazole group of free histidine has a pK₂ of 6.0. Use the…arrow_forward
- Biology: The Dynamic Science (MindTap Course List)BiologyISBN:9781305389892Author:Peter J. Russell, Paul E. Hertz, Beverly McMillanPublisher:Cengage Learning