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J Proteome Res. 2015 Apr 3;14(4):1645-56. doi: 10.1021/acs.jproteome.5b00089. Epub 2015 Mar 18.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

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†Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States.
‡Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
§Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States.


Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.


2D gel electrophoresis; DEAE Affigel Blue column chromatography; extracellular GAPDH; multimer; serum GAPDH

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