A binding site for divalent metal ions on the ATPase from Halobacterium saccharovorum is proposed. This site is different
from the catalytic site which binds ATP and a complexed divalent metal ion. ...Occupation of the second site greatly stimulates
the rate of ATP hydrolysis and the affinity of the catalytic site for the metal ion-ATP complex. The time-dependent inhibition
of the ATPase, which occurs during catalysis and which is known to be caused by the retention of ADP, is also dependent on
the occupation of this metal ion binding site. The binding of the metal ion apparently induces extremely tight binding of
ADP after the departure of Pi. Mg2+, Mn2+, Zn2+, Co2+, and Ca2+ were tested and showed both the activating and the inhibitory
effects, although their binding constants for ATP and the second metal ion binding site were quite different. The characteristic
shapes of the nonlinear ATP hydrolysis curves obtained with different metal ions, and different ratios of metal ion and ATP,
could be explained with the established dissociation constants. On this basis, a model for the ATPase was developed with two
catalytic cycles: one in which the second metal ion binding site is occupied, and another in which it is empty. These pathways
are connected by metal ion-dependent equilibria.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
For F1-ATPases from mitochondria and chloroplasts, tight binding of Mg2+ and ADP without Pi at a catalytic site had been reported
as a cause of enzyme inhibition. The time dependence of this ...inhibition and the effect of various agents on this process have
been described (Du, Z., and Boyer, P. D. (1990) Biochemistry 29, 402-407, and references therein). Similar results are now
reported for the ATPase from Halobacterium saccharovorum. The nonlinear hydrolysis kinetics were modulated by nitrate, azide,
sulfite, GTP, ADP in the absence of ATP, or Pi in characteristic ways, in good analogy with the effects of these agents on
F1 enzymes. The similarity to the F1 systems suggests that it is tight ADP binding that is affected. Although these reactions
of the H. saccharovorum ATPase occurred on different time and concentration scales than those of F1-ATPases, the two systems
do not appear to be fundamentally different. The hydrolytic mechanism of the H. saccharovorum ATPase thus identifies this
enzyme as a member of the F0F1-ATPase family.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is ...characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.
Nucleotide‐binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of α‐32PATP. A high‐affinity site, ...located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross‐linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP‐inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non‐catalytic nucleotide‐binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1‐ATPase which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The (ν
+ ν
) combination band of water has been investigated in aqueous solutions of proline, glycinebetaine and glycerol and in a three component solution with each of these substances and albumin. ...It is shown that the hydrogen bonding strength between the water protons and proline or betaine is higher than between water and glycerol. Betaine exhibits a higher affinity versus the oxygen of water than does proline.
The water binding capacity of pure proline solutions is unchanged in a three-component solution with albumin. Proline neither enhances nor reduces the solubility of this highly soluble protein. In contrast, in a three-component solution with betaine, the solubility of both betaine and album in is reduced. It is assumed that these solute particles compete for the same binding position on the water molecule. Concentrated glycerol solutions with very low water concentrations dissolve a considerable amount of albumin, which points to the fact that the protein must be partly dissolved in glycerol itself.
In this study, the authors examine the effects of chloride and protons on the chromophores of halorhodopsin and bacteriorhodopsin, with the expectation that these ions, substrates of the two ...light-driven pumps, respectively, will interact with the apoproteins sufficiently to perturb the retinal moiety. All experiments were with cell envelope vesicles at high salt concentrations, which preserved transport activity, i.e., either in 4 M NaCl or in 1.5 M Na sub(2)SO sub(4) with added NaCl. They find that in contrast with the photocycle of bacteriorhodopsin, whose major kinetic component shows sharp pH dependence, the flash-induced absorbance changes in halorhodopsin are virtually independent of pH between 5 and 9. Chloride, on the other hand, which does not affect bacteriorhodopsin under these conditions, has profound effects on the photocycle of halorhodopsin: after addition of chloride, the flash yield for halorhodopsin is greatly enhanced, and the time for recovery from flash bleaching is increased about 4-fold.
In a light-dependent reaction (3.5 kilolux) at pH 5, the evolution of hexanal, ethane, and ethylene has been established with cell suspensions of the diatom,
Phaeodactylum tricornutum.
During this ...process, chlorophyll and carotenoids are partially bleached. Addition of 25 millimolar α-linolenic acid or 12 millimolar docosahexaenoic acid yield total pigment destruction and enhancement of ethylene and ethane formation (by about 150 and 7,600%, respectively), whereas hexanal production decreases by 70%. Eicosapentaenoic acid, the major polyunsaturated fatty acid in diatoms, stimulates both ethane and hexanal formation (by about 1,400 and 130%, respectively), but reduces ethylene production (by about 60%). This competition suggests that the production of the volatile compounds is closely connected, although hexanal and ethylene obviously possess different unsaturated fatty acids as precursors. Both the kind of the fatty acids and their relative amounts seem to determine the pattern of the evolved hydrocarbons. The presence of 10 millimolar propylgallate inhibits the evolution of the volatile compounds by about 80%, indicating that radical formation might play a key role in this light-dependent cascade of reactions.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The influence of different anions on the deprotonation of the retinal Schiff base of halorhodopsin in the dark was investigated. We find that a large number of anions cause a significant increase of ...the pKa of the Schiff base, an effect attributed to binding to "site I" on the protein. The concentration dependencies of the spectroscopic shifts associated with the changes of the pKa yielded dissociation constants (and thus binding energies) for the anions, which were related to the Stokes radii. The data fit the predictions of electrostatic interaction between the anions and the positive charge associated with site I, if the latter is located within a few angstroms from the surface of the protein. The specificity of site I toward various anions is quantitatively explained by the differences in the change of Born energy upon transfer of the anions from water to the binding site. The changes in the deprotonation energy of the Schiff base upon the binding of anions, delta delta Gdeprot, could be calculated from the delta pKa at infinite anion concentration. Unexpectedly, the delta delta Gdeprot values were remarkably close to the energies of binding to site I. Thus, site I and the Schiff base are strongly electrostatically coupled, either because of close proximity or because of the possibility of allosteric energy transfer between them.
The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of
Phaeodactylum tricornutum have been studied with spin labels using 0.2 M external Ni
2+ to obtain spectra solely from ...labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50–60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3–8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5–2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.
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