BabcockHall

Insulin stimulates the rates of transcription of the genes for hexokinase II and hexokinase IV (also known as glucokinase). See table 15-5 in Nelson and Cox, Principles of Biochemistry, 6th edition.

I wonder whether the peak at 2.09 ppm might be acetone. Some people clean their NMR tubes with acetone, and so seeing a little residual peak from it is not impossible.

My guess is that the 1.43 ppm peak is water in the deuterated chloroform (which is unlikely to be completely dry), but I don't see a residual HCCl₃ peak near 7.26 ppm. Did you use CDCl₃ as your NMR solvent? If you had some ICHD₂ it would show up as a 1:2:3:2:1 pentet. A C-13 spectrum might not be a bad idea, especially if your present sample is at sufficient concentration.

One problem that we face in this type of question is that insulin increases the rates both of processes that lead to glucose 6-phosphate production and also of processes that lead to its consumption. The steady-state level of glucose 6-phosphate will be affected by both processes.

Insulin raises the amount of a particular class of GLUT transporter found on the plasma membrane of muscle and adipose tissues. I am not sure what you last sentence means.

The question is huge. A proper answer could be found by reading specific portions of several chapters in a standard biochemistry textbook.

I agree with CharonY. For many common antibacterial solutions, there are tables that provide concentration ranges at which the compound is typically used. These tables may be found in reference books such as Sambrook et al. However meropenem is unfamiliar to me, and I don't recall having seen it in any table with which I am familiar; therefore, some of these tables may not be what your need.

Exotoxins are released from the cell, but endotoxins are not. Endotoxins are either on the cell surface or are intracellular.

I could be wrong about this, but perhaps the chemical shift is concentration-dependent. As the concentration of water in the chloroform increases, there is more opportunity for hydrogen bonding. I don't have a citation handy, however.

Good afternoon,

We have a been working on a phosphatase, which gives indifferent over expression levels in E. coli using a T7-based system. We also have a triple mutant variant which also does not express well. Both proteins are trickier to purify than other proteins with which I have worked, because they seem prone to precipitation. We have not yet found conditions which reliably keep them in solution at concentrations above roughly 2 mg/mL. More recently we were given a plasmid with the phosphatase as a GST fusion, and it over expresses well. We are wondering what is likely to be the best way to obtain a gene for our triple mutant as a GST fusion. We see two basic alternatives:

A. site-directed mutagenesis of the GST fusion we already have.

B. recloning of the triple mutant that we now have in to a GST-based plasmid.

We are in the process of obtaining the sequence of the triple mutant but do not have it at the moment. I don't have any prior experience cloning into systems using affinity tags (my cloning skills in general are quite limited and out of date). I have consulted Amersham's GST fusion handbook (specifically Chapter 2). However, it is not very specific about how to obtain a plasmid with the insert in the correct reading from.

Does anyone have an opinion on which strategy is likely to be better? Does anyone have a good resource for information on cloning into GST fusion plasmids. Thank you for your time.

It is difficult to be certain what your question is. Is the enzyme a phosphatase? Many phosphatases take para-nitrophenylphosphate as a substrate and produce para-nitrophenol as a product. Is the para-nitrophenylphosphate dissolved in 1 mM HCl. Or do you mean that the para-nitrophenol is dissolved in 1 mM HCl?

What do you know about the acid-base chemistry of para-nitrophenol? What do you know about the UV/VIS spectrum of this compound?

I don't think that S_N2 dehydration is clear terminology. E2 dehydration makes more sense if we are discussing an elimination.

I think that when sodium hydride accepts a proton, the side products will be Na⁺,Br^- and H₂ gas. I see no reason why an epoxide could not form in d.

What makes you think that spider's web protein is water soluble? With what does ninhydrin react?

There are many examples of how hormones (and also certain intracellular signals) regulate oppositely directed enzymes in a reciprocal manner. The most obvious examples are phosphofructokinase-1 and fructose 1,6-bisphosphatase and how fatty acid biosynthesis is regulated at its first committed step in eukarotic organisms. The paired enzymes of glycogen anabolism and catabolism are also well-studied.

I am not sure what you mean by "example of fatty acids entering TCA from anabolic reaction." Fatty acids do not enter the TCA cycle from an anabolic reaction; they enter after beta-oxidation, which is catabolic. Do you think that the question wishes you to discuss the joint regulation of anabolic and catabolic pathways?

It is my understanding that proton transfer may be, but need not be, concerted with the bond breaking or bond forming between heavy atoms. However the reading I have done so far has not addressed this point explicitly.

If an aqueous reaction speeds up as the concentration of protons is increased, then it is subject to specific acid catalysis. If a reaction speeds up as the concentration of a buffering species is increased at constant pH, then general acid (or general base) catalysis may be occurring (but nucleophilic catalysis would need to be ruled out before one could conclude that it was IIUC). The key is that if one raises the concentration of a buffer then one does not change the pH (to a very good first approximation), yet the reaction speeds up. Therefore, the weak acid or weak base component of the buffer must be the catalytic species.

William P. Jencks' Catalysis in Chemistry and Enzymology has a good discussion (all of Chapter 3), despite its being an older book. Many enzymes use a histidyl residue to perform general acid or general base catalysis; these include chymotrypsin and ribonuclease A. General acid and general base catalysis may be more important for enzymes than in organic synthesis, because much biochemistry takes place near neutral pH, where both protons and hydroxide ion are in very low concentration.

If the nitrogen were protonated, it would not have a lone pair of electrons to coordinate to the iron ion.

That looks fine to me.

That's better. The third from the last structure is a six-membered ring with a positive charge, and there is no reason to draw it looking so distorted. I don't understand what is happening between the third-from-last and the second to last structure. Finally, a base (which could be water) is needed to accept a proton to form the alkene product. The water molecule you drew is doing something that I do not understand.

When the electrons of the sigma bond leave the carbon atoms, that carbon atom should have a formal positive charge, which I do not see in your drawing.

I suggest drawing the first carbocation in the reaction and asking what kind of carbocation it is (primary, secondary, or tertiary). Carbocation rearrangements occur when an electron pair from a sigma bond moves to form a different bond. They are often seen when they lead to a carbocation of greater stability in the product than in the reactant.

Have you learned anything about how carbocations can rearrange?

I understand that Thiamin can become depleted in alcohol abuse since it is used in the metabolism of alcohol to convert Pyruvate to Acetyl-CoA. However, I believe that Vitamins B2, B3 and B5 are also somehow involved, so why is it that these vitamins don't also become depleted and require supplementation?

 

 

Alcohol may be oxidized to acetyl CoA, but pyruvate is not on this pathway. Also, coenzymes such as thiamine are not consumed in enzymatic reactions.