Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a very versatile molecule with a variety of functions in living systems. According to information from the University of Missouri, mammalian GAPDH helps in microtube bundling, membrane fusion, export of nuclear RNA, phosphotransferase activity, DNA repair, DNA replication and programmed death of neuronal cells among others. However, what it is widely known for is its glycolytic function where it converts D-glyceraldehide-3-phosphate into 1,3-biphosphoglycerate. GAPDH has the ability to recognize a wide variety of molecules.
GAPDH and Metabolism
The enzyme helps in breaking down glucose to provide carbon molecules and energy by catalyzing the sixth step of glycolysis. This step is important in the generation of carbon molecules and energy. In the oxidation process, electrons are transferred from NAD+ to NADH, which is the first step in the tertiary stage of glycolysis. During this step, aldehyde is also converted into carboxylic acid.
The process leads to the release of energy used in the second step where inorganic phosphate molecule is moved to the GAP intermediate, creating 1,3-biphosphoglycerate, which has a high potential for the transfer of phosphoryl. The enzyme reduces the positive activation energy through the application of general base catalysis and covalent catalysis. The reaction involves a number of complex processes.
Oxidative reactions generate energy that is harvested for metabolism, which is why some scientists describe GAPDH as the “essence of metabolism.” It also helps in the conservation of energy.
All glycolysis steps occur within the cytosol, which is also where the reactions that GAPDH catalyze also take place. Red blood cell studies have shown that GAPDH and various glycolytic enzymes make complexes within cell membranes. When glycolytic enzymes are in close proximity, the speed at which glucose is broken down increases significantly.
The enzyme is also involved in a number of non-metabolic processes like the initiation of apoptosis and transcription activation. The transcription link was discovered in 2003 when researchers found that GAPDH was present in OCA-S transcriptional coactivator complex, which also contains lactate dehydrogenase. In the past, scientists believed that these two proteins were only concerned with metabolism. GAPDH may help in linking the transcription of genes to the metabolic state.
One of its important functions is signaling oxidative stress induced cell death. Under some conditions, the enzyme may prevent the apoptosis of cells, deflecting them instead to autophagy.
Other studies have shown that GAPDH helps in initiating apoptosis when it binds to DNA. It binds to ubiquitin ligase protein known as Siah1. The protein then initiates controlled cell death by targeting nuclear proteins for degradation. Deprenyl helps to reduce GAPDH’s apoptotic action by preventing S-nitrosylation.
In the presence of oxidative stress, the enzyme may function as a reversible metabolic switch. Cells exposed to oxidants require large quantities of antioxidant cofactor NADPH. GAPDH may become inactive when oxidants treatments are used, leading to a temporary re-routing of the metabolic flux to Pentose Phosphate Pathway. This gives cells the chance to produce more NADPH. Some antioxidant systems require more NADPH during stress conditions. NADPH is also useful in recycling gluthathione.
Another area where GAPDH is involved is transport of vesicle from endoplasmic reticulum to the Golgi. This is part of the route taken by secreted proteins. The enzyme also plays an important role in neurodegenerative diseases, including prostate cancer. Other studies have also shown that GAPDH’s activity increases when it is exposed to physiological concentrations of insulin in 3T3 cells.
GAPDH functions as a housekeeping gene that plays an important role in fully characterized systems. The enzyme is normally abundant in all cells where it usually helps in normalizing protein.
GAPDH antibody can be used to control the loading of protein in Western blots. It is useful for immunohistochemistry-Parrafin and peptide ELISA. The GAPDH antibody also helps in visualizing cells by fluorescence microscopy.
Several things may influence appropriate concentrations, including the concentration of antigen, secondary affinity to antibody, length of incubation and temperature among others.