ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.     

Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria.

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.

2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.

3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.

4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-³²P_i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.

5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.

6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.

7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD⁺ by succinate.

8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Positive supercoiling is generated in the presence of Escherichia coli SeqA protein

In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo. The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.     

Ultrasound indentation of bovine knee articular cartilage in situ

We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges. In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks (n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E(dyn)), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly (r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E(dyn), ultrasound reflection coefficient of articular surface (R) and thickness, respectively. E(dyn) and R were site dependent (p<0.05, Kruskall-Wallis H test). E(dyn) was significantly higher (p<0.05, Kruskall-Wallis Post Hoc test) in LPG (mean+/-SD: 10.1+/-3.1MPa) than in MTP (2.9+/-1.4MPa). In FMC, E(dyn) was 4.6+/-1.3MPa. R was significantly (p<0.05) lower at MTP (2.0+/-0.7%) than at other sites (FMC: 4.2+/-0.9%; LPG: 4.4+/-0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E(dyn) (r=0.678, p<0.01) and especially with the equilibrium Young's modulus (reference device, r=0.874, p<0.01) but it was not associated with R (r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E(dyn) and R provides valuable diagnostic information on cartilage status.     

Biomechanical properties of knee articular cartilage

Structure and properties of knee articular cartilage are adapted to stresses exposed on it during physiological activities. In this study, we describe site- and depth-dependence of the biomechanical properties of bovine knee articular cartilage. We also investigate the effects of tissue structure and composition on the biomechanical parameters as well as characterize experimentally and numerically the compression-tension nonlinearity of the cartilage matrix. In vitro mechano-optical measurements of articular cartilage in unconfined compression geometry are conducted to obtain material parameters, such as thickness, Young's and aggregate modulus or Poisson's ratio of the tissue. The experimental results revealed significant site- and depth-dependent variations in recorded parameters. After enzymatic modification of matrix collagen or proteoglycans our results show that collagen primarily controls the dynamic tissue response while proteoglycans affect more the static properties. Experimental measurements in compression and tension suggest a nonlinear compression-tension behavior of articular cartilage in the direction perpendicular to articular surface. Fibril reinforced poroelastic finite element model was used to capture the experimentally found compression-tension nonlinearity of articular cartilage.     

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.