Many thanks in advance!! -I manage to recreate the glycolytic pathway in vitro u

Question

Many thanks in advance!!

-I manage to recreate the glycolytic pathway in vitro using only purified components. I start the pathway by adding ATP that is radiolabeled using radioactive Phosphorus to the in vitro reaction. MOST LIKELY, predict all the molecules that will get radiolabeled when the pathway reaches equilibrium (assuming I am not replenishing the NAD+ present in the reaction). Explain your answer.

-A deficiency in Biotin could cause a decrease in the liver’s ability to carry out Gluconeogenesis. Is this TRUE / FALSE? Explain your answer.

- A certain protein, “X” is normally found in the lysosome. I make two versions of X in vitro. One is glycosylated with Mannose-6-PO4 and the other is not. I introduce these two versions of X into the cytoplasm of cells, and follow their localization within the cell. MOST LIKELY predict where the two versions will end up in the cell. Explain.

- I normally purify a protein from a mixture using an affinity column containing a lectin. This column cannot purify the protein if the mixture is first treated with a glycosidase. If I take the protein mixture and heat it up to first denature the proteins, MOST LIKELY will I still be able to purify my protein using this column (YES/NO)? MOST LIKELY what will the measured specific activity of the protein in this case be (HIGH/MEDIUM/LOW/NONE)? Explain your answer.

Explanation / Answer

Answer:

Answering 2 and 3rd question.

2. A deficiency in Biotin could cause a decrease in the liver’s ability to carry out Gluconeogenesis. Is this TRUE / FALSE? Explain your answer.

Answer: True, explanation: In gluconeogenesis glucose is synthesized from non-carbohydrate precursor’s pyruvate, lactate, oxaloacetate, glycerol and amino acids. Liver is the main site of gluconeogenesis. Pyruvate carboxylase enzyme is involved in the carboxylation of pyruvate leading to the formation of oxaloacetate.

Pyruvate carboxylase consists of 4 identical subunits and each subunit contains a single biotin molecule. Pyruvate carboxylase utilizes biotin as a prosthetic group that is covalently attached to a lysine residue. Biotin also serves as a carrier of activated CO2.

Since biotin plays a critical role in the activity of Pyruvate carboxylase which is a key enzyme in the gluconeogenesis pathway, therefore a deficiency in biotin could cause a decrease in the livers ability to carry out gluconeogenesis,

3. Proteins are first sorted in the Trans Golgi network based on their final destination. Lysosomal proteins carry a unique marker in the form of mannose-6-phosphate (M6P) groups and are recognized by a transmembrane receptor protein present in the trans golgi network. Thus the protein which is glycosylated with mannose-6 phosphate will bind to mannose-6-phosphate receptor (M6PR) in golgi and will be transported to lysosomes.

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