\vec H^ + /2\bar e Stoichiometry of the NADH:Ubiquinone Reductase Reaction Catalyzed by Submitochondrial Particles

Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q₁ gives the value of n = 4. Thermally induced deactivation of Complex I [1,2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q₁-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q₁-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Catalytic properties of mitochondrial NADH-ubiquinone reductase (Complex I)

Qualitative and quantitative characteristics of the reactions catalyzed by the most complex and least understood proton translocating unit of the mammalian respiratory chain (NADH-ubiquinone oxidoreductase, Complex I) are described for enzyme preparations differing in degree of resolution--from intact mitochondria to homogeneous small enzyme fragments. Special attention is given to the problems and pitfalls of reliable interpretation of the kinetic analysis of the enzyme activities. Detailed analysis of the problems concerning the slow active/inactive reversible enzyme transition is provided.     

Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites

The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki = 40 microM) than with APAD+ (Ki = 150 microM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.     

The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

The structure of the rough-type lipopolysaccharide from Shewanella oneidensis MR-1, containing 8-amino-8-deoxy-Kdo and an open-chain form of 2-acetamido-2-deoxy-D-galactose

The LPS from Shewanella oneidensis strain MR-1 was analysed by chemical methods and by NMR spectroscopy and mass spectrometry. The LPS contained no polysaccharide O-chain, and its carbohydrate backbone had the following structure: (1S)-GalNAco-(1-->4,6)-alpha-Gal-(1-->6)-alpha-Gal-(1-->3)-alpha-Gal-(1-P-3)-alpha-DDHep-(1-->5)-alpha-8-aminoKdo4R-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P, where R is P or EtNPP. There are several novel aspects to this LPS. It contains a novel linking unit between the core polysaccharide and lipid A moieties, namely 8-amino-3,8-dideoxy-D-manno-octulosonic acid (8-aminoKdo) and a residue of 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine, GalNAco) in an open-chain form, linked as cyclic acetal to O-4 and O-6 of D-galactopyranose. The structure contains a phosphodiester linkage between the alpha-D-galactopyranose and D-glycero-D-manno-heptose (DDHep) residues.     

Relationship of hepatocyte ploidy levels with body size and growth rate in mammals.

To elucidate possible causes of the elevation of genome number in somatic cells, hepatocyte ploidy levels were measured cytofluorimetrically and related to the organismal parameters (body size, postnatal growth rate, and postnatal development type) in 53 mammalian species. Metabolic scope (ratio of maximal metabolic rate to basal metabolic rate) was also included in 23 species. Body masses ranged 10(5) times, and growth rate more than 30 times. Postnatal growth rate was found to have the strongest effect on the hepatocyte ploidy. At a fixed body mass the growth rate closely correlates (partial correlation analysis) with the cell ploidy level (r = 0.85, P < 10(-6)), whereas at a fixed growth rate body mass correlates poorly with ploidy level (r = -0.38, P < 0.01). The mature young (precocial mammals) of the species have, on average, a higher cell ploidy level than the immature-born (altricial) animals. However, the relationship between precocity of young and cell ploidy levels disappears when the influences of growth rate and body mass are removed. Interspecies variability of the hepatocyte ploidy levels may be explained by different levels of competition between the processes of proliferation and differentiation in cells. In turn, the animal differences in the levels of this competition are due to differences in growth rate. A high negative correlation between the hepatocyte ploidy level and the metabolic scope indicates a low safety margin of organs with a high number of polyploid cells. This fact allows us to challenge a common opinion that increasing ploidy enhances the functional capability of cells or is necessary for cell differentiation. Somatic polyploidy can be considered a "cheap" solution of growth problems that appear when an organ is working at the limit of its capabilities.     

Structure of the core part of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21

The core-lipid A region of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21 was studied using NMR spectroscopy, ESI MS, and chemical analysis after alkaline deacylation, deamination, and mild-acid hydrolysis of the lipopolysaccharides. The following general structure of the major core oligosaccharides is proposed: [abstract: see text] where all sugars are in the pyranose form and have the D configuration unless otherwise stated, Hep and DDHep=L-glycero- and D-glycero-D-manno-heptose, respectively, K=H, and Q=H in strain 8 or alpha-Glc in strains 7, 14, 15, and 21. In addition, several minor structural variants are present, including those lacking Ara4N in strains 7 and 15 and having the alpha-GlcN residue N-acylated to a various degree with glycine in strains 7, 8, 14, and 21. In strain 14, there are also core oligosaccharides with K=amide of beta-D-GalpA with putrescine, spermidine, or 4-azaheptane-1,7-diamine; remarkably, these structural variants lack either the PEtN group or the alpha-Hep-(1-->2)-alpha-DDHep disaccharide fragment at alpha-D-GalpA. While structural features of the inner core part are shared by Proteus strains studied earlier, the outermost Q-(1-->4)-alpha-GalNAc-(1-->2)-alpha-DDHep-(1-->6)-alpha-GlcN oligosaccharide unit has not been hitherto reported.