BabcockHall

A student whom I know wishes to do some DNA melting experiments, following them by UV/VIS spectrophotometry. What sort of solvent should she use for a recirculating bath? I have used ethylene glycol/water for low temperature work, but I have not done much high temperature work. If anyone knew of a Methods article on this subject, please feel free to pass along the reference.

About 15 minutes of sunshine on one's face and hands is sufficient for production of vitamin D3.

Ef-Tu is the protein involved in codon/anticodon recognition, and this spends one GTP per peptide bond. Ef-G is responsible for translocation of the ribosome, and it also hydrolyzes GTP. With respect to mRNA synthesis, look at the reaction catalyzed by RNA polymerase, and see if that helps.

Try looking in the index for the three steps of protein synthesis. Or try looking up recognition of the anticodon and translocation of the ribosome.

That guess is incorrect. The reaction between an amino acid and its cognate tRNA requires ATP as a substrate, and the reaction produces AMP and the charged tRNA. The third product is pyrophosphate, and its fate is to be hydrolyzed to two molecules of inorganic phosphate. What textbook are you using? If you think that GTP is not involved in protein synthesis (later in the process than the tRNA syntheses), then you need a better source of information on protein synthesis than whatever you are using now.

How many high energy bonds are spent to produce a charge tRNA (an aminoacyl tRNA)? During the three steps of protein synthesis, are any additional high-energy bonds spent in the form of GTP?

3PG is not the same thing as glycerol phosphate. Draw out the structures and assign oxidation numbers to carbon, and the difference becomes obvious.

Good idea. A long time ago, I immobilized alkaline phosphatase myself, but I doubt that it would be worth my time to learn how to immobilize thrombin. Better to go with the commercial product. Any thoughts about which is better, removal of thrombin via a benzamidine column versus immobilized thrombin?

Can you explain your reasoning? I suggest you consider tRNA synthetases when totaling up the number of high-energy bonds that must be spent. I am also not sold on your calculation of number of high energy bonds for mRNA, but perhaps we can worry about proteins first, then RNA.

Thanks. We were planning to do classical (low pressure) cation exchange, owing to a large difference in theoretical pI values between the two proteins, thrombin and the N-terminal and middle domains from FliM, which was well expressed. I imagine if this fails, we will go with a column of benzamidine or possibly biotin-labeled thrombin. We are now routinely checking proteins with mass spectrometry. This is our first attempt at preparing this construct; therefore, we do not yet have HPLC conditions worked out.

Each peptide bond costs four high-energy phosphate bonds, assuming that the correct codon-anticodon interaction is selected every time.

Good Afternoon, We would like to remove thrombin from a post-nickel column digest. However, our protein and thrombin have similar molecular weights; therefore, I don't hold out much hope for gel filtration. We think that cation exchange may work, inasmuch as our protein has a low pI. We are interested in co-crystallization experiments with a second protein. There is a benzamidine column, but it is a little pricey. There is also biotin-labeled thrombin, but it has the same drawback. There is also APMSF as an alternative to PMSF, and this would at least inactivate the thrombin. Thanks for any suggestions.

I agree with CharonY. I might focus on one to three kinds of reactions. Transaminations (catalyzed by PLP-containing aminotransferases) are responsible for the synthesis of several nonessential amino acids (Ala, Glu, Asp, and a precursor to Ser come to mind). The reactions that produce Gln from Glu and which produce Asn from Asp are similar but not identical. Both involve ATP, but the ATP is cleaved differently, and the intermediates are somewhat different.

Many monophosphate esters have a pH rate maximum near 4 for non enzymatic hydrolysis. Hydrolysis at 100 °C is pretty rapid, but obviously much slower at room temperature. At pH 7, it is even slower, but it is still not zero. Also microbial degradation is a problem, unless you are working with sterile solutions.

Do you need to know the number of high energy bonds for the synthesis of mRNA or for the protein?

I am not as familiar with platinum as I am with palladium as a reduction catalyst. However, in McMurry's textbook, there is a reaction which shows an unsaturated ketone is reduced to a ketone in the presence of Pd and H2. In other words, you can find conditions that would reduce the carbon-carbon double bond without reducing the ketone. A ketone would be more easily reduced using sodium borohydride.

Experimental error should not be categorically ruled out. Enzyme assays may be run outside the limits of where they respond linearly to the concentration of enzyme, for instance. If you calculated the total number of units by finding the specific activity and multiplying by the number of milligrams of protein, then the protein assays are another potential source of error.

You are asking a large and somewhat nebulous question. It takes at least four high energy bonds to make one peptide bond. However, given the existence of proofreading by Ef-Tu in E. coli, it sometimes takes five. DNA polymerase also engages in proofreading, which costs energy.

Evans7, There is at least one error in your thinking, and perhaps more than one. Enzymes and cofactors (coenzymes or metal ions) are catalysts that don't change the equilibrium constants of the reactions with which they are associated. Of the reactions in glycolysis that do not consume or produce ATP, some have favorable and some have unfavorable values of deltaG°'.

I am not a medical doctor. If one has a serious abdominal injury, my understanding is that presumably nonpathogenic strains of E. coli are released into areas that they ordinarily are not found and that this is dangerous. Offhand, I don't know of other similar examples, but it would not surprise me if they exist.

One exothermic is not the same as exergonic. The former deals with enthalpy, and the latter deals with Gibbs' free energy. The hydrolysis of ATP is thermodynamically favorable, but the reasons are both enthalpic and entropic. Two, you are asking two questions. The first is about mechanical work, and the second is about active transport. It is also worth pointing out that not all active transport requires ATP, but some does. Three, when you say "structural change" are you referring to the enzyme which is utilizing ATP or to something else. It also seems to me that your question might be about the general phenomenon of biological coupling, or it might be very specific, such as a question about the catalytic cycle of the actin•myosin complex. Perhaps you can help us by providing some context.

You may be thinking of the Maillard reaction.

@OP, When an alcohol reacts with an anhydride, the general reaction is R'OH + RC(O)-O-C(O)R ==> R'-O-C(O)R + RC(O)OH. The main product is an ester, and one equivalent of an acid is the side product. This is a pretty generous hint toward balancing the equation, and it goes along with John Cuthber's hint.

@OP, It was unclear to me how you balanced this equation. Did you use some sort of a program? I suggest balancing the equation yourself and show us what you have. Without a properly balanced equation, the limiting reagent calculations are bound to fail.

I suggest that you write a balanced equation first. Whatever your equation balancer is, I don't think it is helping you in this instance.