Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic l-alanine:4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial l-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of l-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial l-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial l-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial l-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of l-alanine:4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment

Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle

Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitotic nuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process

Spectrum Sharing For Information Freshness: A Repeated Games Perspective

We consider selfish sources that send updates to a monitor over a shared wireless access. The sources would like to minimize the age of their information at the monitor. Our goal is to devise strategies that incentivize such sources to use the shared spectrum cooperatively. Earlier work has modeled such a setting using a non-cooperative one-shot game, played over a single access slot, and has shown that under certain access settings the dominant strategy of each source is to transmit in any slot, resulting in packet collisions between the sources' transmissions and causing all of them to be decoded in error at the monitor. We capture the interaction of the sources over an infinitely many medium access slots using infinitely repeated games. We investigate strategies that enable cooperation resulting in an efficient use of the wireless access, while disincentivizing any source from unilaterally deviating from the strategy. Formally, we are interested in strategies that are a subgame perfect Nash equilibrium (SPNE). We begin by investigating the properties of the one-stage (slot) optimal and access-fair correlated strategies. We then consider their many-slot variants, the age-fair and access-fair strategies, in the infinitely repeated game model. We prove that the access-fair and age-fair strategies are SPNEs for when collision slots are longer than successful transmission slots. Otherwise, neither is a SPNE. We end with simulations that shed light on a possible SPNE for the latter case.Comment: Accepted for publication in IEEE Global Communications Conference (GLOBECOM) 202

Shuttling components of nuclear import machinery involved in nuclear translocation of steroid receptors exit nucleus via exportin-1/CRM-1* independent pathway

AbstractThe nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general ‘nuclear import factors’ that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s)

Gradation of Nanoparticle Size by Stokes' Law: A New Approach for Synthesis of CdS Nanoparticles

The synthesis technique and its allied process parameters have a specific effect on the nucleation, growth-dominated microstructure and properties of nanostructure materials. The properties of semiconductor nanoparticles strongly depend on its size, shape, composition, crystallinity and structure. Recently, semiconductor nanoparticles have been extensively investigated and gained much interest due to their unique properties and applications in diverse areas of science and technology. A new controlled technique for synthesis of CdS nanopartlicles by means of kinetic approach using well-known Stokes' law for free body falling in quiescent and viscous fluid has been employed. Nanoparticles of cadmium sulfide (CdS) have been synthesized by simple controlled chemical method using IR radiation heating without using any capping agent and stirring. The desired concentration of aqueous solutions of cadmium chloride (CdCl2.2H2O) and thioacetamide (CH3CSNH2) were reacted in a controlled manner by IR radiation heating at the reaction area (top layer of reactants solution) of solution results the formation of CdS nanoparticles following Stokes' law. The as-synthesized nanoparticles were characterized by XRD, optical spectroscopy and SEM with EDX analysis

Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs