Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.

Metallothionein 3 (MT-3), also known as growth inhibitory factor (GIF), exhibits a neuroinhibitory activity. Our lab and others have previously shown that this biological activity involves interacting protein partners in the brain. However, nothing specific is yet known about which of these interactions is responsible for the GIF activity. In this paper, we are reporting upon new proteins found interacting with MT-3 as determined through immunoaffinity chromatography and mass spectrometry. These new partner proteins—Exo84p, 14-3-3 Zeta, α and β Enolase, Aldolase C, Malate dehydrogenase, ATP synthase, and Pyruvate kinase—along with those previously identified have now been classified into three functional groups: transport and signaling, chaperoning and scaffolding, and glycolytic metabolism. When viewed together, these interactions support a proposed model for the regulation of the GIF activity of MT-3.

Microglial cells play a major role in host defense of the central nervous system. Once activated, several functional properties are up-regulated including migration, phagocytosis, and secretion of inflammatory mediators such as cytokines and chemokines. Little, if anything, is known about the metabolic changes that occur during the activation process. High-resolution 1H nuclear magnetic resonance spectra obtained from perchloric acid extracts of human microglial cell cultures exposed to lipopolysaccharide (LPS) or morphine were used to both identify and quantify the metabolites. We found that human microglia exposed to LPS had increased concentrations of glutamate and lactate, whereas the cells exposed to morphine had decreased concentrations in creatinine, taurine, and thymine. Glutamate and creatinine were the key metabolites differentiating between the two stimuli. These results are discussed in terms of activation and differences in the inflammatory response of human microglial cells to LPS and morphine.

A HPLC-fluorescence method, using the fluorophore SBD-F (ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate), was adapted for the quantification of metallothioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. The method was first optimized using a rabbit liver metallothionein. The effects of EDTA, tris(2-carboxyethyl)phosphine, and SBD-F on the labeling efficiency were studied. The optimized method was then applied to evaluate the amount of metallothionein in the mussels either exposed to cadmium in the laboratory or collected from the Casablanca coast, Morocco. The concentrations of metallothioneins measured in the field samples describe the degree of contamination of the sites and are reflected by distinct isoform patterns.