The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG)

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.     

Synthesis and Chiral Recognition of Nickel(II) Macrocyclic Complex with (R)‐Naphthylethyleneamine Pendant Groups and its Self‐Assembled Framework

A novel nickel(II) hexaaza macrocyclic complex, [Ni(L^R,R)](ClO₄)₂ ( 1 ), containing chiral pendant groups was synthesized by an efficient one‐pot template condensation and characterized (L^R,R═1,8‐di((R)‐α‐methylnaphthyl)‐1,3,6,8,10,13‐hexaazacyclotetradecane). The crystal structure of compound 1 was determined by single‐crystal X‐ray analysis. The complex was found to have a square‐planar coordination environment for the nickel(II) ion. Open framework [Ni(L^R,R)]₃[C₆H₃(COO)₃]₂ ( 2 ) was constructed from the self‐assembly of compound 1 with deprotonated 1,3,5‐benzenetricarboxylic acid, BTC^3?. Chiral discrimination of rac‐1,1′‐bi‐2‐naphthol and rac‐2,2,2‐trifluoro‐1‐(9‐anthryl)ethanol was performed to determine the chiral recognition ability of the chiral complex ( 1 ) and its self‐assembled framework ( 2 ). Binaphthol showed a good chiral discrimination on the framework ( 2 ). The optimum experimental conditions for the chiral discrimination were examined by changing the weight ratio between the macrocyclic complex 1 or self‐assembled framework 2 and racemates. The detailed synthetic procedures, spectroscopic data including single‐crystal X‐ray analysis, and the results of the chiral recognition for the compounds are described. Chirality, 25:54‐58, 2013 © 2012 Wiley Periodicals, Inc.     

A Functional Analysis of the Yeast Ubiquitin Ligase Rsp5: The Involvement of the Ubiquitin-Conjugating Enzyme Ubc4 and Poly-Ubiquitination in Ethanol-Induced Down-Regulation of Targeted Proteins

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae. We have found that the Ala401Glu rsp5 mutant is hypersensitive to various stresses, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. The ubiquitination and the subsequent degradation of stress-induced misfolded proteins are indispensable for cell survival under stress conditions. In this study, we analyzed the ubiquitin-conjugating enzyme Ubc4 and the poly-ubiquitination of targeted proteins involved in the function of Rsp5 under ethanol stress conditions. Ubc4 was found to be important in yeast cell growth and poly-ubiquitination of the bulk proteins in the presence of ethanol. The general amino acid permease Gap1 is poly-ubiquitinated via Lys63 and is down-regulated after the addition of ammonium ions through a process requiring Rsp5. We found that Gap1 was removed from the plasma membrane in the presence of ethanol in a Rsp5-dependent manner, and that the disappearance of Gap1 required Ubc4 and involved the lysine residues of ubiquitin. Our results also indicate that Lys6 of ubiquitin might inhibit the disappearance of Gap1. These results suggest that Rsp5 down-regulates the ethanol-induced misfolded forms of Gap1. In addition, it appears that the substrates of Rsp5 are appropriately poly-ubiquitinated via different lysine residues of ubiquitin under various growth conditions.     

The amino acid permease AAP8 is important for early seed development in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The development of seeds depends on the import of carbohydrates and amino acids supplied by the maternal tissue via the phloem. Several amino acid transporters have been reported to be expressed during seed and silique development in Arabidopsis thaliana (L.) Heynh. Here we show that mutants lacking the high affinity amino acid permease 8 (At1g10010) display a severe seed phenotype. The overall number of seeds and the number of normally developed seed is reduced by ∼50% in siliques of the Ataap8 T-DNA insertion mutant. This result could be reproduced in plants where expression of AtAAP8 is targeted with an RNAi approach. The seed phenotype is correlated with a specifically altered amino acid composition of young siliques. Aspartic acid and glutamic acid are significantly reduced in young siliques of the mutants. In correlation with the fact that AAP8 is a high affinity transporter for acidic amino acids, translocation of ¹⁴C-labelled aspartate fed via the root system to seeds of the mutants is reduced. AAP8 plays a crucial role for the uptake of amino acids into the endosperm and supplying the developing embryo with amino acids during early embryogenesis.     

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21–-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.     

Reduced cellular Ca availability enhances TDP-43 cleavage by apoptotic caspases

Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca²⁺ availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca²⁺, affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca²⁺ availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15 kDa, in addition to the well-known fragments of 35 and 25 kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.