In 1956, E. P. Kennedy and S. B. Weiss published their study of membrane lipid phosphatidylcholine (lecithin) synthesis in rat liver. Their hypothesis was that phosphocholine joined with some cellular component to yield lecithin. In an earlier experiment, incubating 32 P-labeled phosphocholine at physiological temperature (37 °C) with broken cells from rat liver yielded labeled lecithin. This became their assay for the enzymes involved in lecithin synthesis. Determine the optimal pH for this enzyme and characterize the enzyme activity at different pH values. -O-P-O-CH2-CH₁₂-N(CH3)3 Phosphocholine H₂C-O-C-R HC-O-C-R2 + + + Cell fraction + ? HC-O-P-O-CH₁₂-CH₂-N(CH), O Phosphatidylcholine The researchers then centrifuged the broken cell preparation to separate the membranes from the soluble proteins. They tested three preparations: whole extract, membranes, and soluble proteins. Table 1 summarizes the results. Table 1: Cell fraction requirement for incorporation of 32p-phosphocholine into phosphatidylcholine Preparatio 32 P-Phosphocholine incorporated Tube num ber n into lecithin Whole 1 6.3 μmol extract 2 Membranes 18.5 μmol Soluble prot 3 eins 2.6 μmol 4 | Was the enzyme responsible for this reaction a soluble protein or a membrane-bound enzyme? Why? options: a mixture of membrane-bound enzyme Soluble protein has all the enzymes required to generate lecithin ↳ a membrane-bound enzyme, ble the preparation in tube a had the largest amount of p p-lecithin ↳ a soluble protein, blc less 33 p-labeled phosprocholine was required to generate lecithin in tube 3 Having determined the location of the enzyme, the researchers investigated the role of pH on enzyme activity. They carried out their standard assay in solutions buffered at different pH values between 6 and 9. The graph shows the results. The enzyme activity is the amount, in nanomoles per liter, of 32P-phosphocholine incorporated into phosphatidylcholine. optimal pH: Which statement best describes the enzyme activity illustrated by the graph? options: It is about 33% lower at pH 8 than at pH 6. ↳ It is lower at more basic, or higner, pH values. is about 15% greater at pH 8 than at pH 6. Reactions with phosphorylated intermediates commonly require a divalent metal ion. The researchers tested Ca²+, Mn2+, and Mg2+to determine if a divalent metal ion was important in this reaction. The graph shows the results. Mg2+ Enzyme activity (nanomoles per liter of incorporated 32P-phosphocholine) 88 9 5 Mn2+ Ca2+ 10 15 20 Metal ion concentration (μM) | Which statements describe the metal ion dependence of the system? ☐ Calcium activates the system at any concentration. Magnesium activates the system when present at concentrations above 5 μM. A low concentration of manganese, 5-10 μM, activates the system. Magnesium inhibits the system at concentrations between 5-15 μM. The researchers reasoned that the reaction might require energy. To test the hypothesis, they incubated rat liver membranes and 32P-phosphocholine with different nucleotides. Because the ATP sold in 1956 was not as highly purified as modern commercial preparations, the researchers used two different ATP sources, lot 116 and lot 122. Table 2 gives the results. Table 2: Requirement of nucleotides for phosphatidylcholine synthesis from phosphocholine Tube number Which statements do the results shown in Table 2 support? ATP speeds the reaction if CTP is present. UTP can substitute for CTP in the reaction. ☐ The reaction requires CTP. ☐ ATP from lot 116 was not pure. Enzyme activity (nanomoles per liter of incorporated 32P-phosphocholine) 10- 8 1 5 6 7 8 9 pH Nucleotide added 32p incorporated into lecithin 1 5 μmol ATP from lot 116 5.1 μmol 2 5 μmol ATP from lot 122 0.2 μmol 5 μmol ATP from lot 122 نيا 3 0.4 μmol +0.5 μmol GDP 5 μmol ATP from lot 122 4 15.0 μmol +0.5 μmol CTP 5 μmol ATP from lot 122 5 10.0 μmol +0.1 μmol CTP 5 μmol ATP from lot 122 6 0.4 μmol +0.5 μmol UTP 7 0.5 μmol CTP with no ATP 8.0 μmol Close 4 UTP GDP جا LAT LCTP Phosphocholine+ 4 membrane-bound enzyme 4soluble proteins ↳ Mpat ↳ Mga+ Complete the equation for the reaction they studied. ↳ Ca2+ →phosphatidylcholine