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INSTRUCTIONS: 1. Donotopenthisexamuntilinstructedtodoso. 2. Thisisaclosedbookexam.WewillobservetheCODEOFACADEMICINTEGRITY. Pleasesignthepledgebelow. 3. Writeyournameoneverypage. 4. WriteyouranswersinPEN,notpencil,unlessyouarepreparedtowaiveyour righttomakearegraderequest. 5. Makesureyouanswerallofthequestions. 6. PleasewriteLEGIBLY! 7. Notethatcorruptionofacorrectanswerwitherroneousorsuperfluoustextwill resultinlesscredit. 8. Pleaseenteryouranswerstotheshortanswerquestionsinthespaceprovided donotwriteonthefacepageortheothersideofthepage. 9. Time:youhave50minutestocompletethisexam. IpledgemyhonorthatIhaveupheldtheCODEOFACADEMICINTEGRITYoftheUniversity ofPennsylvaniaincompletingthisexamination
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(4 pts)
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5) Humanbloodserumcontainsaclassofenzymesknownasacidphosphatases whichhydrolyzeorganicphosphatesunderslightlyacidicconditions(pH5.0): ROPO32+H2OROH+HPO42 Acidphosphatasesareproducedbyawiderangeofcellsincludingerythrocytes, andthoseoftheliver,kidney,spleenandprostategland.Theenzymeproducedby theprostateglandisofclinicaldiagnosticvaluebecauseanincreaseinitslevelsin thebloodcanbeindicativeofprostatecancer.Thephosphatasefromtheprostate glandisstronglyinhibitedbytartrateanion(adicarboxylicacid)butacid phosphatasesfromothercelltypesarenot.Howmightthisinformationbe exploitedforthedevelopmentofaspecificprocedureformeasuringtheactivity prostaticacidphosphataseactivityinhumanbloodserum?Ifyouweretouseits catalyticactivityasanindexoftheamountofthisenzymeinbloodserumwhat precautionswouldyouhavetotake?
(8 pts)
The activity of the prostatic acid phosphatase would correspond to the total acid phosphatase activity of the blood sample minus the acid phosphatase activity measured in the presence of a sufficiently high concentration of tartrate to completely inhibit the prostatic enzyme. If catalytic activity is to be a reliable index of the amount of the enzyme in blood serum it is not only crucial that sufficient tartrate is added to the sample when enumerating the contribution of the prostatic enzyme to total acid phosphatase activity but that the concentration of substrate (an organic phosphate) added is sufficient to ensure that the enzyme is operating at close to maximal activity (Vmax) such that reaction velocity, v, is directly proportional to prostatic enzyme concentration: v = k[E]t where k is a proportionality constant and [E]t is the total concentration of the prostatic enzyme.
6) Youhaveidentifiedanorganismthatcanmaintainthefluidityofitsplasma membraneevenatverylowtemperatures.Youanalyzeplasmamembrane samplesfromthisorganismaftergrowthatmoderatetemperaturesandafter growthatlowtemperaturesanddeterminetheircontentsofsaturatedandcis unsaturatedphospholipids.Whatwouldyouexpecttofind? A. Nochangeintheratioofsaturatedtounsaturatedphospholipidsatlow temperatures B. Anincreaseintheratioofsaturatedtounsaturatedphospholipidsatlow temperatures. C. Anincreaseintheratioofunsaturatedtosaturatedphospholipidsatlow temperatures Howwouldyourexpectationschange,ifatall,iftheunsaturatedphospholipidsin questionwereinthetransratherthanthecisform?Brieflyexplainyourreasoning.
(4 pts) (3 pts)
cis double bonds in the fatty acyl hydrocarbon chains of phosphopholipids introduce
kinks that decrease the ease with which phospholipids associate with each other to form a crystal lattice (undergo solidification) at lower temperatures. trans double bonds do not have this effect because they do not introduce kinks into the fatty acyl hydrocarbon chains; their behavior is more like that of fatty acyl substituents possessing saturated hydrocarbon chains.
NAME:
7) TheuptakeofDglucosefromtheintestinallumen(fromthecontentsofthegut) bytheNa+coupledtransporterassociatedwiththeluminal(apical)membraneof thecellsoftheintestinalepithelium: A. isanexampleofantiport. B. iscapableofmediatingtheaccumulationofglucoseevenwhen [Na+]extracellular=[Na+]intracellularprovidingthemembranepotentialisnegative (insidenegative). C. iscapableofmediatingtheaccumulationofglucoseevenwhenthe membranepotentialispositive(insidepositive)providingthat [Na+]extracellular=[Na+]intracellular. D. isonlycapableofmediatingtheaccumulationofglucosewhenthe extracellularconcentrationofNa+isgreaterthantheintracellular concentration.
(4 pts)
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(4 pts)
Whichofthefollowingstatementsaboutallostericenzymesandtheirregulation is/areCORRECT A. Theyareoftenfoundatkeystepsinmetabolicpathwaysandundergo feedbackregulation. B. Theyusuallyexhibitcooperativekinetics. C. Thebindingofanallostericactivatorincreasestheactivityoftheenzyme atallsubstrateconcentrations. D. Allostericactivatorsusuallyincreasetheconcentrationofsubstrate requiredfor50%saturationoftheenzymebyenhancingthesigmoidicity ofvversus[S]plots. E. Theeffectsofactivatorsandinhibitorsonthecooperativeinteractions betweensubstratebindingsubunitspermitchangesinvevenwhenthe concentrationofsubstrateremainsthesame. F. Ifacompetitiveinhibitorisaddedtoasolutioncontaininganallosteric enzyme,theratioofenzymemoleculesintheactiveconformationto thoseintheinactiveconformationwould(probably)increase.
9) Fromwhatyouknowoftheenergyyieldfromtheanaerobicversusaerobic metabolismofglucose,whatwouldyoupredictforacellthatiscapableof operatinganaerobicallyoraerobicallywhenthecellistransferredfromanaerobic toaerobicconditions(ifthecellistoharvestthesameamountofenergyperunit timeunderbothconditions)?Assumethatthesupplyofglucoseisplentiful: A. Agreaterthan16foldincreaseintherateofglucoseconsumption. B. Anapproximately2to4foldincreaseintherateofglucoseconsumption. C. Agreaterthan16folddecreaseintherateofglucoseconsumption. D. Anapproximately2to4folddecreaseintherateofglucose consumption. E. Nochangeintherateofglucoseconsumption.
(4 pts)
NAME:
11)Supposethatyouhavebeengivenasuspensionofbovineheartmitochondriathat ismediatingthesteadystateoxidationofanNADcoupledsubstrateinamedium containinganexcessofoxidizablesubstrate,NAD+,ADP,Pi,andO2.Whatwould youexpecttoobserveifanuncoupler,forexample2,4dinitrophenol(2,4DNP), wereaddedtothissuspension? A. Adecreaseintherateofsubstrateoxidation. B. Anincreaseintherateofsubstrateoxidation. C. AdecreaseintherateofNADHoxidation. D. AnincreaseintherateofNADHoxidation. E. AdecreaseintherateofATPsynthesisfromADPandPi. F. AnincreaseintherateofATPsynthesisfromADPandPi. G. AdecreaseintherateofO2consumption. H. AnincreaseintherateofO2consumption. (4 pts)
SHORTANSWERQUESTIONS ONLYAFEWSENTENCESATMOSTARENEEDEDFORACOMPLETEANSWERTO EACHSECTION Pleaseenteryouranswersinthespaceprovidedtrytoavoidwritingonthefacepage ortheothersideofthepage. 12) Allostericenzymesaremadeupofmultiplesubunitsandsomeoftheseenzymes existinonlytwoconformationalstates,designatedRorT(forRelaxedand Tense).Assumeforthetetramericallostericenzymeinquestionthatsubstrate (S)onlybindstotheRstate.Intheabsenceofligand(substrate,allosteric inhibitorsorallostericactivators),thetwostates(RorT)ofsuchanallosteric proteinareinequilibrium:RT.TheequilibriumconstantL(definedasL=T/R) isusuallyverylarge;thatis,theamountoftheproteinintheTstateismuch greaterthantheamountintheRconformation. ExplainwhathappenstotheequilibriumbetweenRandTasincreasingamountsof substrateareaddedtoasolutioncontainingtheenzyme. (5 pts)
The equilibrium between the T and R conformational states would be displaced in favor of the R conformation as increasing amounts of substrate are added to the solution containing enzyme. If the enzyme is in dynamic equilibrium between the T and R states and substrate only binds to the R state, the addition of increasing amounts of substrate to the solution containing enzyme would serve to trap increasing amounts of the enzyme in the R state.
NAME:
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13)
OnthebasisoftheseresultswhatisthelikelymechanismofDglucoseuptakeby thissystem?Assumethat:
v(Dglucose)=Vmax[Dglucose] Km+[Dglucose] where,v(Dglucose)=initialrateofDglucoseuptake Vmax=maximalrateofDglucoseuptake Km=concentrationofDglucosewhenv=Vmax/2
InwhatwaydoesDgalactoseappeartoinfluencethekineticsofDglucose uptake?Explainyourreasoning.
(5 pts) The addition of 10 mM D-galactose to the uptake medium does not change the magnitude of the 1/v(D-glucose) intercept. Given that the kinetics of D-glucose uptake approximate Michaelis-Menten kinetics (yield a straight line relationship when 1/v(Dglucose) is plotted against 1/[D-glucose], this implies that D-galactose does not decrease or increase the maximal capacity of the transporter. When 1/[D-glucose] = 0, 1/v(D-glucose) = 1/Vmax; v(D-glucose) = Vmax. However, D-galactose does increase Km. When 1/v(D-glucose) = 0, Km/Vmax[D-glucose] = -1/Vmax and 1/[D-glucose] = -1/Km. In words, the addition of 10 mM D-galactose to the uptake medium results in a less negative value for -1/Km, meaning that the absolute value of Km is increased. Dgalactose therefore appears to be a simple competitive inhibitor of D-glucose transport. That the transport of D-glucose is both saturable and stereospecific in so far as D-galactose competes with D-glucose for uptake, implies that the transport observed is carrier-mediated.
NAME:
Wouldthetransportpathwayyouhaveidentifiedbecapableofmediatingthe accumulationofDglucosebymousetumorcells?Explainyourreasoning.
(5 pts) No. Facilitated diffusion simply serves to accelerate equilibrative transport. It cannot establish a concentration gradient because it is freely reversible and not susceptible to primary or secondary energization by factors that impose directionality on the transporter.
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NAME:
14) BondswhosehydrolysisproceedswithlargenegativevaluesofG(customarily morenegativethan6kcal/mol;25kJ/mol)areoftenreferredtoas"highenergy bonds".Itisimportanttonote,however,thatnoneofthebondsofATPhave especiallyunusualproperties.Theterm"highenergybond"(sometimes denotedasADP~P)whenappliedtothiscompoundcanthereforebemisleading. ExplainandcommentonATPsstatusnotsomuchasaveryhighenergy phosphatebutmoreasanintermediateenergyphosphate.Whatbearing doesthishaveonourunderstandingoftheprimingreactionsintheenergy investmentphaseofglycolysisandthesubstratelevelphosphorylationsinthe energygenerationphase?
(12 pts)
The phosphate bonds of ATP are not particularly strong bonds. Instead, the "high energy" character of ATP derives, in part, from its charge distribution. In the ATP molecule all three phosphate bonds are negatively charged. These "like charges" are crowded together, and their repulsion contributes to the instability of this region of the ATP molecule. ADP, on the other hand, is much more stable. In the physiological pH range, ATP has three to four negative charges whose mutual electrostatic repulsion is partially relieved by its hydrolysis to ADP. The crucial role of ATP perhaps has less to do with its status as a high energy phosphate but more to do with its status as an intermediate energy phosphate. An important feature of the ATP-ADP system is that it is a primary connecting link between high- and low-energy phosphate compounds. ATP is of intermediate phosphate transferring potential. ADP can accept a phosphate group from a compound with a higher free energy of hydrolysis, for instance 1,3bisphosphoglycerate (1,3-bisPG) and phosphoenolpyruvate (PEP) in the energygeneration phase of glycolysis by substrate-level phosphorylation, and the ATP so formed can then donate it to an acceptor molecule to yield a phosphate compound lower on the scale, for instance the phosphorylated sugars glucose-6-phosphate (G6P) and fructose-1,6-bisphosphate (F16bP) generated in the priming reactions of the energy-investment phase of glycolysis. For this to happen (to be thermodynamically spontaneous) it is imperative that the free energy of hydrolysis of ATP is less than those of 1,3-bisPG and PEP and higher than that of G6P and F16bP.
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