Glyceraldehyde-3-phosphate dehydrogenase, known as GAPDH, is one of the most common proteins found inside nearly all cells. Its high abundance reflects its fundamental role in maintaining basic cellular functions necessary for survival. In the laboratory, “GAPDH control” refers to its use as a standard reference point for experiments. Understanding how this protein is regulated is essential for correctly interpreting scientific data that uses it as a benchmark.
The Primary Role of GAPDH
The original and most recognized function of GAPDH is its participation in glycolysis, the metabolic pathway that breaks down glucose for energy. It acts as a glycolytic enzyme, catalyzing the sixth step of the process by oxidizing an aldehyde group. Specifically, GAPDH converts glyceraldehyde-3-phosphate (\(text{G3P}\)) into 1,3-bisphosphoglycerate (1,3-BPG). This reaction is coupled with the reduction of the electron carrier nicotinamide adenine dinucleotide (\(text{NAD}^+\)) to \(text{NADH}\).
The \(text{NADH}\) generated by GAPDH is later shuttled to the mitochondria to fuel the electron transport chain, which produces the vast majority of the cell’s adenosine triphosphate (\(text{ATP}\)). Because glycolysis is a foundational process required by almost every living cell to generate \(text{ATP}\), the enzyme must be highly active and abundant. This constant, high demand for energy generation is the reason why GAPDH is expressed at high concentrations in nearly all tissues.
Why It Is Used as an Experimental Standard
GAPDH’s consistently high expression level across many cell types led to its adoption as a “housekeeping gene” in molecular biology. These genes are required for the maintenance of basic cellular function, and their expression is theoretically constant under normal conditions. Scientists rely on this assumption of consistency to normalize their experimental results.
In techniques like Western blotting, which measures protein levels, researchers use GAPDH as a “loading control.” This involves measuring the amount of GAPDH in each sample to confirm that an equal amount of total protein was loaded into the gel before separation. By comparing the signal of a target protein to the unchanging signal of GAPDH, differences observed can be attributed to the experimental treatment rather than to errors in sample preparation.
A similar standardization approach is used in quantitative polymerase chain reaction (\(text{qPCR}\)), a technique for measuring gene expression at the messenger \(text{RNA}\) (\(text{mRNA}\)) level. GAPDH \(text{mRNA}\) levels serve as a reference to calculate the relative abundance of a target gene’s \(text{mRNA}\). The final result is expressed as a ratio, correcting for variations in the amount of starting material or differences in the efficiency of the \(text{cDNA}\) synthesis step.
When Its Expression Varies
While GAPDH’s consistency under standard laboratory conditions is generally accepted, its expression is far from universal. This variability means that using it as a control can sometimes introduce significant error, potentially invalidating experimental conclusions. One major condition affecting GAPDH levels is hypoxia, or low oxygen availability, which is common in many disease states.
When cells experience hypoxia, the transcription of the GAPDH gene is often upregulated, producing more \(text{mRNA}\) and protein. This response maximizes the efficiency of anaerobic glycolysis, a process that continues without oxygen to generate minimal \(text{ATP}\). Because many experimental models involve tissues that become hypoxic, such as in stroke or heart disease studies, GAPDH levels can fluctuate dramatically and unpredictably.
Metabolic stress, which occurs when nutrient availability is limited, can also significantly alter the enzyme’s expression and activity. GAPDH levels are also highly variable across different tissue types and developmental stages, such as during cellular differentiation. Specific types of cancer cells, which rely heavily on glycolysis for rapid growth (known as the Warburg effect), often exhibit much higher GAPDH expression compared to surrounding healthy tissue.
Post-translational modifications (\(text{PTMs}\)) also affect the enzyme’s stability and function. For instance, processes like S-nitrosylation or phosphorylation can alter the enzyme’s structure, causing it to become inactive in glycolysis and potentially relocate within the cell to perform non-glycolytic tasks. When GAPDH is used as a control in studies involving these conditions, it acts more like a regulated experimental variable than a stable reference point. Researchers must validate GAPDH’s stability for every unique cell type and stress condition before relying on it for accurate normalization.
Functions Beyond Energy Production
The enzyme’s unreliability as a universal control is partly explained by its involvement in roles entirely separate from its glycolytic function. Under specific conditions of cellular stress, GAPDH shifts its localization and function within the cell. This change in role represents a form of cellular control, where the protein is regulated to serve a different purpose, such as protecting the cell from damage.
One notable non-glycolytic role is its participation in DNA repair and maintenance of genomic integrity. Upon certain types of DNA damage, GAPDH can rapidly translocate from the cytoplasm into the nucleus. Once inside the nucleus, it interacts with various proteins to help regulate gene expression and facilitate the repair process, effectively switching from an energy generator to a nuclear regulator.
GAPDH also plays a part in programmed cell death, or apoptosis. When a cell receives a strong apoptotic signal, the enzyme can be chemically modified, causing it to bind to or move towards the cell membrane or mitochondria to promote the cell’s demise. These stress-induced roles, which also include membrane fusion and regulation of vesicle transport, confirm that GAPDH is a highly versatile and regulated enzyme, making its use as an invariant experimental standard highly context-dependent.