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.     

Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation — FH469→Stop

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469_Trp(TGG). The GA mutation changes this codon into the amber stop codon, and is referred to as FH_469Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH_469Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.     

Ultrastructure of myocardial widened Z bands and endocardial cells in two teleostean species

Summary Widened myocardial Z bands and endocardial cells are described in two teleostean species Cichlasoma meeki and Corydoras aeneus. Widened Z bands containing mainly amorphous and electron-dense material were seen in a number of myocardial cells. Further, similar material may occur in large amounts beneath the sarcolemma and at intercellular junctions. Occasionally, we observed continuity between the latter material and that in expanded Z bands.In C. meeki the ventricular endocardial layer consists of two structurally different cell types, whereas in C. aeneus only one cell type was seen. The functional aspects of widened Z bands are discussed.     

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.

The growth of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> as influenced by CO<Subscript>2</Subscript>, light,and flue gases

In order to achieve recognition as environmentally friendly production, flue gases should be used as a CO₂ source for growing the microalgae Chlorella sorokiniana when used for hydrogen production. Flue gases from a waste incinerator and from a silicomanganese smelter were used. Before testing the flue gases, the algae were grown in a laboratory at 0.04, 1.3, 5.9, and 11.0 % (v/v) pure CO₂ gas mixed with fresh air. After 5 days of growth, the dry biomass per liter algal culture reached its maximum at 6.1 % CO₂. A second experiment was conducted in the laboratory at 6.2 % CO₂ at photon flux densities (PFD) of 100, 230, and 320 μmol photons m^?2 s^?1. After 4 days of growth, increasing the PFD increased the biomass production by 67 and 108 % at the two highest PFD levels, as compared with the lowest PFD. A bioreactor system containing nine daylight-exposed tubes and nine artificial light-exposed tubes was installed on the roof of the waste incinerator. The effect of undiluted flue gas (10.7 % CO₂, 35.8 ppm NO _x , and 38.6 ppm SO₂), flue gas diluted with fresh air to give 4.2 % CO₂ concentration, and 5.0 % pure CO₂ gas was studied in daylight (21.4?±?9.6 mol photons m^?2 day^?1 PAR, day length 12.0 h) and at 135 μmol photons m^?2 s^?1 artificial light given 24 h day^?1 (11.7?±?0.0 mol photons m^?2 day^?1 PAR). After 4 days’ growth, the biomass production was the same in the two flue gas concentrations and the 5 % pure CO₂ gas control. The biomass production was also the same in daylight and artificial light, which meant that, in artificial light, the light use efficiency was about twice that of daylight. The starch concentration of the algae was unaffected by the light level and CO₂ concentration in the laboratory experiments (2.5–4.0 % of the dry weight). The flue gas concentration had no effect on starch concentration, while the starch concentration increased from about 1.5 % to about 6.0 % when the light source changed from artificial light to daylight. The flue gas from the silicomanganese smelter was characterized by a high CO₂ concentration (about 17 % v/v), low oxygen concentration (about 4 %), about 100 ppm NO _x , and 1 ppm SO₂. The biomass production using flue gas significantly increased as compared with about 5 % pure CO₂ gas, which was similar to the biomass produced at a CO₂ concentration of 10–20 % mixed with N₂. Thus, the enhanced biomass production seemed to be related to the low oxygen concentration rather than to the very high CO₂ concentration.     

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.     

Uptake of foreign ferritin in platy Xiphophorus maculatus (Poeciliidae: Teleostei)

The ability and capacity of various tissues in platy Xiphophorus maculatus L. to take up horse-spleen ferritin injected into the blood stream are described. Ferritin was injected intraperioneally, and the cellular uptake was demonstrated as Prussian blue precipitations in tissues treated with acid ferrocyanide solutions. Ferritin was detected within the heart endocardial cells and macrophages in the trunk kidney and spleen, 1/4 h after the injection, i.e. foreign ferritin was taken up very rapidly by these cells. When the time elapsed between the ferritin injection and sacrifice exceeded 6 h, these cells, and also macrophages in the gill and intestine, were almost completely filled with ferritin. At these stages, however, the amounts of Prussian blue precipitations per volume unit of the tissue were much larger in the heart than in the other organs studied in the present work, i.e. the endocardial tissue seems to play an important role in the clearance of the blood circulation in this species. We suggest that this tissue in platy is specialized to endocytose waste and foreign macromolecules, including pathogenic particles, from the blood stream. The eosinophilic and neutrophilic granulocytes do not appear to take up foreign ferritin, i.e. these cells may play no endocytotic role in the clearance of foreign macromolecules in platy.     

Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.     

BOX elements modulate gene expression in Streptococcus pneumoniae: impact on the fine-tuning of competence development

More than 100 BOX elements are randomly distributed in intergenic regions of the pneumococcal genome. Here we demonstrate that these elements can affect expression of neighboring genes and present evidence that they are mobile. Together, our findings show that BOX elements enhance genetic diversity and genomic plasticity in Streptococcus pneumoniae.     

Properties and biological role of streptococcal fratricins

Competence for natural genetic transformation is widespread in the genus Streptococcus. The current view is that all streptococcal species possess this property. In addition to the proteins required for DNA uptake and recombination, competent streptococci secrete muralytic enzymes termed fratricins. Since the synthesis and secretion of these cell wall-degrading enzymes are always coupled to competence development in streptococci, fratricins are believed to carry out an important function associated with natural transformation. This minireview summarizes what is known about the properties of fratricins and discusses their possible biological roles in streptococcal transformation.