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2,3-Butandione 2-monoxime (BDM) is a widely used myosin inhibitor with an unclear mode of action. In this report, we investigated the mechanism of BDM oxime group nucleophilic reactivity on the phosphoester bond of ATP. BDM increased the ATPase activity of skeletal myosin subfragment 1 (S1) under conditions in which ATP cleavage is the rate-limiting step (K+, EDTA-ATPase activity of native S1 and Mg2+-ATPase activity of trinitrophenylated S1 and partially unfolded S1). Furthermore, the effect of BDM on the S1-bound adenosine 5′-(β,γ-imido) triphosphate (AMPPNP) 31P NMR spectrum suggests that BDM changes the microenvironment around the phosphorus atoms of myosin-bound nucleotide. A computational search for the BDM-binding site in the adenosine 5′-[γ-thio] triphosphate (myosin–ATPγS) complex predicted that BDM is located adjacent to the nucleotide on myosin. Therefore, we propose that the BDM oxime group catalytically assists in ATP cleavage, thereby enhancing the ATPase activity of myosin in a manner analogous to pralidoxime-mediated reactivation of organophosphate-inactivated acetylcholinesterase. This is the first study suggesting that oxime provides catalytic assistance for ATP cleavage by an ATP-hydrolyzing enzyme.

Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

To identify novel obesity-related genes in adipose tissue, differential display was performed using bombesin receptor subtype-3 (BRS-3)-deficient mice. These mice exhibit mild late-onset obesity. We report that a gene, Urb, is upregulated in these mice. Full-length Urb cDNA is approximately 3 kb long and comprises an open reading frame of 949 amino acid residues. Interestingly, Urb mRNA expression in brown adipose tissue of BRS-3-deficient mice is fourfold higher than that in wild-type controls. Enhanced Urb mRNA expression was also observed in brain, digestive tissues, kidney, and lung. Within the brain, Urb mRNA is detected in the dorsal endopiriform nucleus and choroid plexus. A T31 radiation hybrid mapping panel revealed that the Urb gene maps to mouse chromosome 16. Collectively, these findings suggest that Urb may have a unique function in the regulation of body weight and energy metabolism.

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. We have isolated a novel mouse gene for ubiquitin carboxyl-terminal hydrolase isozyme L4. The gene named Uchl4 encodes a novel member of the family of ubiquitin carboxyl-terminal hydrolases (UCHs) whose predicted amino acid sequence shows 95% identity to mouse UCH-L3 and 94% identity to human UCH-L3. Genomic structure, chromosome localization, and expression pattern of Uchl3 and Uchl4 were characterized in the mouse. Both Uchl3 and Uchl4 were expressed in various tissues examined; however, expression level was quite lower in Uchl4. While Uchl3 consists of at least 9 exons spanning about 12 kb, Uchl4 was an intronless gene with a size of about 2 kb. By PCR-based analysis with T31 radiation hybrid mapping panel, Uchl3 and Uchl4 were mapped on mouse chromosome 9 and 14, respectively.

Hepatocyte growth factor (HGF) specifically activates Met tyrosine kinase receptor, leading to mitogenic, motogenic, and morphogenic responses in a wide variety of cells. To know a role of HGF in Xenopus embryogenesis, loss-of-function mutation was introduced by dominant expression of truncated tyrosine kinase-negative Met. When tyrosine kinase-negative Met mRNA was micro-injected into two-cell to eight-cell stages Xenopus embryos, the liver development was mostly impaired and structures of pronephros and the gut were grossly underdeveloped in the restricted, late stage of development. These results strongly suggest that functional coupling between HGF and Met is essential for the development of internal organs originated from primitive gut and possibly involved in embryonic skeletogenesis. Together with developmental abnormality in mice mutated with HGF or Met gene, essential role of HGF for liver development is highly conserved from amphibian to mammalian species.

HGF-like protein (HLP), also known as macrophage stimulating protein, is a ligand for Ron receptor tyrosine kinase, a member of the c-Met/hepatocyte growth factor receptor family. We cloned a Xenopus homologue of the Ron/HLP receptor (Xron) and HLP (Xhlp) and analyzed temporal and spatial expressions. Both Xron and Xhlp mRNA were detected in Xenopus embryos at the cleavage stage, as maternally pooled RNA. Zygotic expression of Xron mRNA was seen in early embryos during midblastula and neurula (stage 22), while Xhlp was evident during midblastula and early tadpole (stage 40). Xron mRNA was localized in the most frontal region of the neural fold and peripheral regions of the neural plate, while Xhlp mRNA was seen in midline of the neural plate (notoplate) and the most frontal regions of the neural fold in early neurula. Thus, functional coupling between Ron receptor tyrosine kinase and HLP may have an important role in development of embryonic neural tissue in Xenopus laevis.