Question

Labeling Studies in Isolated Mitochondria Biochemists have often delineated the metabolic pathways of organic compounds by using a radioactively labeled substrate and following the fate of the label. a. How can you determine whether a suspension of isolated mitochondria metabolizes added glucose to \(\mathrm{CO}_{2}\) and \(\mathrm{H}_{2} \mathrm{O}\) ? b. Suppose you add a brief pulse of \(\left[3-{ }^{14} \mathrm{C}\right]\) pyruvate (labeled in the methyl position) to the mitochondria. After one turn of the citric acid cycle, what is the location of the \({ }^{14} \mathrm{C}\) in the oxaloacetate? Explain by tracing the \({ }^{14} \mathrm{C}\) label through the pathway. How many turns of the cycle are required to release all the \(\left[3-{ }^{14} \mathrm{C}\right]\) pyruvate as \(\mathrm{CO}_{2}\) ?

Short answer

Use radiolabeled glucose and monitor CO2 for radioactive emissions. [3-14C] label ends up in succinate after the first cycle and requires about three cycles to release all 14C as CO2.

Step-by-step solution

Determining Glucose Metabolism to CO2 and H2O

To determine if mitochondria metabolize glucose to CO2 and H2O, provide isolated mitochondria with radiolabeled glucose (e.g., glucose labeled with 14C). Allow metabolism to proceed, then collect emitted CO2. Use a detector to measure the radioactivity of CO2. If the CO2 is radioactive, it indicates that the glucose is metabolized to CO2.

Understanding Pyruvate Entry into the Citric Acid Cycle

[3-14C] pyruvate enters the mitochondria and is converted to acetyl-CoA via the pyruvate dehydrogenase complex. During this conversion, one CO2 is released, and the acetyl group enters the citric acid cycle.

Location of 14C in First Cycle of Citric Acid Cycle

In the citric acid cycle, the acetyl group condenses with oxaloacetate to form citrate. The labeled carbon (14C) becomes the methyl group in citrate. In the first complete cycle, the 14C will distribute between the second and third carbon atoms of succinate, corresponding to the second and third carbon atoms of oxaloacetate.

Releasing 14C through Subsequent Cycles

For 14C to oxidize to CO2, the labeled carbon must proceed through the cycle. Initially, the 14C label in oxaloacetate does not decarboxylate until the alpha-ketoglutarate and succinyl-CoA stages. Complete oxidation of the 14C label as CO2 typically requires three cycles. During these cycles, carbon atoms are lost stepwise as CO2.

Key concepts

Metabolic Pathways

Metabolic pathways are a series of chemical reactions that occur within a cell. These reactions are vital for maintaining life as they allow cells to grow, reproduce, maintain their structures, and respond to their environments. Imagine them as roads leading to a destination, with each pathway following a specific route to convert substrates into energy or other necessary compounds.
A classic example of a metabolic pathway is how glucose is metabolized. When cells need energy, glucose undergoes a process of glycolysis, breaking down into pyruvate. This pyruvate can further undergo transformations to produce ATP, the energy currency of the cell. It's a tightly regulated process, ensuring energy needs are met efficiently without wasting resources.
Biochemically, studying these pathways often involves radioactive labeling. By "tagging" molecules with a radioactive element, scientists can trace where they travel within the cell, providing insights into the steps and efficiency of various metabolic processes. This technique is crucial because it allows us to see the molecular journey and understand better how cells function on a more detailed level.

Citric Acid Cycle

The citric acid cycle, also known as the Krebs cycle, is central to cellular respiration. It occurs in the mitochondria, the powerhouse of the cell. Once glycolysis breaks down glucose into pyruvate, the next step is the citric acid cycle, where pyruvate is further broken down, releasing energy stored in its chemical bonds.
During this cycle, pyruvate is converted into acetyl-CoA, which then combines with oxaloacetate to form citrate. Each turn of the cycle processes this citrate through a series of transformations, ultimately regenerating oxaloacetate, and releasing two carbon dioxide molecules. It's like a wheel that continuously turns, processing carbon atoms and releasing significant energy to be captured in the form of ATP, NADH, and FADH2.
Understanding this cycle is crucial for biochemists. Using radioactive labeling, scientists can follow individual carbon atoms through the cycle. This tracking helps identify at which points the carbon atoms are released or transformed, providing a comprehensive map of their journey and showing precisely how metabolic energy is extracted from nutrients.

Mitochondrial Metabolism

Mitochondrial metabolism refers to the set of metabolic processes that occur within mitochondria, the energy transformers of the cell. Their main function is to generate ATP through processes such as the citric acid cycle and oxidative phosphorylation. Think of mitochondria as tiny power plants within cells, converting fuel into usable energy.
Apart from energy production, mitochondria are also involved in other cell processes, including heat production, regulation of the metabolic cycle, and in some cases, programming of cell death. The efficiency of mitochondrial processes is vital for the overall health and functionality of cells, and by extension, the entire organism.
In biochemistry, understanding mitochondrial metabolism can be enhanced through techniques like radioactive labeling. By tracking specific carbon atoms through the mitochondrial processes, researchers gain insight into the path and transformation of substances within this organelle. This detailed understanding is essential for developing new therapeutic strategies for diseases linked to mitochondrial dysfunction, such as metabolic disorders or neurodegenerative diseases.