1. To accomplish the purification, rabbit brain tissue was homogenized and centr
ID: 42226 • Letter: 1
Question
1. To accomplish the purification, rabbit brain tissue was homogenized and centrifuged to remove insoluble material. Next, the soluble preparation was loaded on top of an ATP-Sepharose column. This is an affinity column in which ATP is covalently linked to a polysaccharide bead. The sample is loaded on top of the column, washed with a low-salt buffer, followed by a wash with a high salt buffer. What is the rationale for using this procedure? Draw a diagram of the expected elution profile.
2. Next, the fractions containing PFK-1 activity were applied to a DEAE-Sephadex (anion exchange) column. The column was equilibrated with a pH = 8.2 buffer. The column was eluted with a salt (ammonium sulfate) gradient and the results are shown in Figure 18.1. Using the elution profile as well as the results from SDS-PAGE analysis shown in Figure 18.2, identify which isozyme is found in each of the two peaks. How might the amino acid composition of PFK-1 B differ from that of PFK-1 A and C based on the manner of elution from the DEAE column?
3. Next, the investigators took Fractions 16-26 from the DEAE-Sephadex column, pooled them, and adjusted the pH to 5.0. This preparation was then loaded onto a CM-52 (cation exchange) chromatography column and eluted with a pH gradient. Fractions 30-35 were collected and pooled, as were Fractions 41-50. Identify the peaks in the chromatogram in Figure 18.3, using information in the SDS-PAGE gel. Based on their elution from the cation exchange column, how might the amino acid compositions of these two proteins differ?
4. Write the reaction catalyzed by PFK-1.
5. There are several allosteric effectors that influence the activity of PFK-1 in the cell. What are they? List both activators and inhibitors of the enzyme.
6. The investigators next carried out kinetic studies using their newly purified PFK-1C isozyme. They studied the catalytic behavior of the enzyme in the presence of the metabolites AMP, inorganic pho i sphate (P ) and fructose-2,6-bisphosphate (F-2,6-BP). The results are shown in Figure 18.4. Additional information concerning the three isozymes response to allosteric effectors is presented in Tables 18.1 and 18.2. a. Compare the ability of PFK-1C to catalyze the phosphorylation of fructose-6-phosphate in the ab i sence of, and in the presence of AMP, F-2,6-BP or P . How do these allosteric effectors influence the velocity of the reaction? b. Evaluate whether the investigators have shown that PFK-1 C is different from the PFK-1 A and PFK-1 B isozymes. Speculate why there might be functional differences among the isozymes.
Explanation / Answer
1. IMAGE IS UPLOADED FOR ELUTION PROFILING ,
4. reaction catalyzed by PFK-1.;
it will converts fructose 6 phosphate +atp to fructose 1,6 bisphosphate +adp
5. both activators and inhibitors of the enzyme.;
PFK1 is a allosteric enzyme , and performs the commiting step in glycolysis , it has many activators and inhibiotrs , THE ENZYME HAS ATP BINDING SITE . IT HAS TWO STATES R STATE AND T STATE . IN R STATE THE ACTIVATORS WILL BIND AND IN T STATE THE INHIBHITORS WILL BIND
ACTIVATORS ; AMP , ADP
INHIBITORS INCLUDE ; PEP ,ATP
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