Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis.

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.     

Urea-hydrolysis-dependent citrulline synthesis by Ureaplasma urealyticum

Some of the ammonia produced by hydrolysis of urea by Ureaplasma urealyticum is channelled into an anabolic pathway with resultant 'de novo' synthesis of citrulline. The organism appears to possess ornithine carbamoyltransferase and carbamoyl phosphate synthetase or some modified form of these enzymes.     

Adenosine 5'-triphosphate synthesis in Escherichia coli submitted to a microsecond electric pulse

The total cytoplasmic ATP content (bound and free) increased in Escherichia coli when the bacteria were submitted to electric pulses with field strengths of 1-6 kV/cm and a decay time of 7-20 microseconds. The electron-transport chain was blocked by cyanide, and ATP synthesis was detected by a luminescence assay. The amount of newly formed ATP depends on the field strength. A total of 150 pmol of ATP was formed per milligram of bacteria submitted to a 3 kV/cm pulse. Synthesis was blocked by uncouplers and ionophores (valinomycin). The F1F0-ATP synthase inhibitor dicyclohexylcarbodiimide blocked a large part of this synthesis. Synthesis was not induced in unc mutants (unc B, unc D). The synthesis of ATP is related to the induced transmembrane potential, not to the Joule heating. A minimum 35-50-mV increase in membrane potential must be maintained for at least 12 microseconds to trigger this synthesis. This very fast energy transduction in bacteria is in good agreement with our previous results concerning submitochondrial particles. Because of the localized character of the induced membrane potential, these results are in agreement with the recent hypothesis of "mosaic proton coupling".     

pH dependence of adenosine 5'-triphosphate synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor

The purified ATP-synthesizing complex from chloroplasts has been reconstituted into phospholipid vesicles with bacteriorhodopsin by use of octyl glucoside. Phosphorylation rates up to 90 mmol of ATP (mg of protein)-1 min-1 have been achieved. The dependence of the steady-state kinetic parameters on external and internal pH for both synthesis and hydrolysis was determined. The Michaelis constants are independent of the magnitude of the pH gradient at external pH values of 6.6 and 8.0. The dependence of the maximum velocity for ATP synthesis on the external pH is bell shaped at a constant pH gradient with a maximum at about pH 6.7. The variation of the maximum velocity with external pH is not dependent on the magnitude of the pH gradient. At external pH values of 6.6 and 8.0, the maximum velocity for ATP synthesis varies with approximately the 2.3 power of the internal hydrogen ion concentration. The maximum velocity for ATP hydrolysis also is dependent on the external pH, with a maximum at about pH 8.4; however, most of the ATPase activity is not coupled to the proton flux. Both Mg2+ and Mn2+ are good cofactors for ATP synthesis and hydrolysis whereas Ca2+ is completely ineffective for synthesis and only about 10% as effective as Mg2+ and Mn2+ for hydrolysis. The results obtained suggest that ATP synthesis or hydrolysis may be coupled to proton pumping indirectly, as, for example, by conformational changes.     

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Calmodulin stimulates both adenosine 5'-triphosphate hydrolysis and synthesis catalyzed by a cardiac calcium ion dependent adenosinetriphosphatase

A Ca2+-dependent ATPase purified from a rabbit heart membrane preparation was compared to the Ca2+-dependent ATPase purified from skeletal muscle sarcoplasmic reticulum. The two ATPases display an identical electrophoretic pattern and an identical Ca2+-concentration dependence. However, only the cardiac preparation exhibits a 2-3-fold activation by calmodulin. This effect is best observed when the molar concentrations of calmodulin and ATPase are equivalent and in the presence of high Ca2+ (approximately 10(-5) M) and ATP (approximately 10(-3) M) concentrations. It is demonstrated for the first time that calmodulin stimulates the rate of ATP synthesis, as revealed by an increased production of Pi and a faster ATP in equilibrium Pi exchange, as well as the rate of ATP hydrolysis. It is also demonstrated that calmodulin activation is expressed with purified and detergent-solubilized enzyme in addition to membrane-bound systems. These findings indicate that the effect of calmodulin is an acceleration of the enzyme turnover, due to direct interaction of calmodulin with the enzyme.     

解脲脲原体诱导脐静脉血管内皮细胞凋亡的研究

目的通过探讨解脲脲原体(Ureaplasma urealyticum,UU)诱导人脐静脉内皮细胞(human umbilicalvein endothelial cells;HUVEC)凋亡的情况,揭示妊娠期间UU垂直传播影响胚胎发育的可能机制。方法不同剂量血清4型UU标准菌株刺激体外培养的HUVEC,通过Annexin-V.FITC/PI双染流式细胞术和DNA Ladder实验观察细胞凋亡情况。结果对照组细胞凋亡率小于各实验组(P<0.01~0.05),其凋亡率和刺激剂量、时间之间呈现一定的剂量-时间-效应关系(P>0.05)。结论UU可诱导人脐静脉内皮细胞凋亡,提示妊娠期间UU感染可能通过诱导脐静脉内皮细胞凋亡破坏胎盘屏障而影响胚胎发育。     

High-density lipoprotein that supports Ureaplasma urealyticum growth.

A high-density lipoprotein with growth-promoting activity for Ureaplasma urealyticum was purified in high yield from equine serum by ammonium sulfate fractionation and molecular filtration. Fractions enriched in growth-promoting activity represented 5% of the total serum protein, and 30 micrograms of the purified protein per ml gave an activity equivalent to that from 100 micrograms of whole serum per ml. The serum was totally replaced by purified lipoprotein when tested in a soy peptone-yeast dialysate or when added to a chemically defined synthetic medium. Polyacrylamide gel electrophoresis indicated that one major protein with growth-promoting activity is present. A total of 10 proteins were distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 75% of the total contributed by two proteins with molecular weights of 160,000 and 170,000. A total of 90% of the lipoprotein was an alpha-protein with a mobility of 0.67 in two-dimensional immunoelectrophoresis (albumin = 1.0). The active component was further characterized as high-density lipoprotein by density ultracentrifugation. Two components with S = 6.4 and S = 15.8 were distinguished by velocity sedimentation. The lipid was removed from lipoprotein during its precipitation with acetone. The growth-promoting activity of delipidized protein was dependent upon the addition of exogenous cholesterol, and [14C]cholesterol was transferred to urea-plasmic cells in cultures containing the delipidized protein. A major portion of the [14C]cholesterol remained associated with the protein during filtration on Sepharose 4B columns.     

Adenosine 5'-triphosphate flux through the north inlet marsh system

The distribution, fluctuation, and short-term transport of total microbial biomass (measured as adenosine 5'-triphosphate [ATP]) was investigated in a large salt marsh creek. Hourly samples were collected synoptically for 25 h from 10 boats positioned across the 320-m width of the creek. Samples were collected from three depths ranging from 0.2 to 8.0 m. Hourly data obtained from each station were graphed, plotting depth against ATP. Subsequently, interpolated ATP values were generated for every one-tenth depth from the surface to the bottom with the use of an 11-point proportional divider. A total of 2,750 values were generated, and a mean value of 0.865 mg of ATP per m was determined. Maximum levels of ATP were found at high tide and minimal values were found at low tide. The distribution of ATP concentrations was found to be complex, with no suggestion of vertical stratification; however, horizontal divisions were apparent. ATP values corrected for direction of flow or velocity indicated two ebb-directed channels; however, when considered in total, there was a net import of ATP through the interface. The total import of ATP for this 25-h sampling period was calculated to be 3.58 kg, corresponding to a net transport of 39.8 mg of ATP per s through the cross section. Results suggest that detailed characterization of a creek transect in terms of ATP or any similar parameter requires the simultaneous measurements of both the concentration of the parameter in question and the velocity at the time and point from which the sample was taken.     

Ureaplasma urealyticum and Ureaplasma parvum in etiology of urogenital mixt-infections

The complex study of 104 vaginal samples from patients with urogenital uroplasmosis was carried out. U. parvum were detected in 67.3% patients, U. urealyticum--in 12.5% and in 20.1% cases--two species were registered at the same time. Isolation of clinical significant concentration of both ureaplasma (> 10(4) CFU/ ml) was detected in about 50% of cases. Expression of inflammation of vaginal mucus depended on the level of concentration of infection agents. U. parvum were associated with bacterial vaginosis, while in urogenital candidosis U. parvum was detected rarer than U. urealyticum. The dominant numbers of clinical ureaplasma were high sensitive to "new" macrolides and chinolons, however the high percent of isolates were resistant to erytromicin and doxiciclin.     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell fractions and enzymatic activities of Ureaplasma urealyticum.

The localization of some enzymic activities in cell fractions of Ureaplasma urealyticum was studied. A quantitative evaluation of the effectiveness of several cell lysis procedures was obtained by using labeled membranes and sucrose density gradient centrifugation. Ultrasonic treatment was found to be the most effective procedure for lysing the cells, whereas digitonin and osmotic shock caused the lysis of only 70 and 50% of the cells, respectively. The localization of selected enzymes in Ureaplasma cells resembled that found in other Mycoplasma species. Adenosine triphosphatase, ribonuclease, deoxyribonuclease, and p-nitrophenylphosphatase activities were located exclusively in the membrane fraction, whereas urease and L-histidine ammonia-lyase were located in the cytoplasm.     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.     

Adenosine 5'-triphosphate is required for the assembly of 11S seed proglobulins in vitro.

Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but mostly monomers were recovered at high translation rates. The prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated that ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated or when ATP was absent suggested that protein misfolding had occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten globular conformations and thereby compete for Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures.     

Catalytic hydrolysis and synthesis of adenosine 5'-triphosphate by stereoisomers of covalently labeled F1-adenosinetriphosphatase and reconstituted submitochondrial particles

Bovine heart F1-adenosinetriphosphatase (F1) was labeled specifically and precisely with 7-chloro-4-nitro-2,1,3-[14C]benzoxadiazole ([14C]NBD-Cl). The stereospecifically labeled F1 (O-beta'-[14C]-NBD-F1) was partially reactivated by LiCl treatment, which could cause rearrangement of the beta subunits to form O-beta', beta'-[14C]NBD-F1. Both labeled enzymes were used to combine with F1-deficient submitochondrial particles (ASU) to form the reconstituted particles O-beta'-NBD-F1-ASU and O-beta', beta'-NBD-F1-ASU, respectively. A comparison of the observed steady-state rates of catalytic ATP hydrolysis and oxidative phosphorylation by these specifically labeled submitochondrial particles (SMP) with those of the unlabeled control samples suggests that oxidative phosphorylation involves more active sites of F1 than catalytic ATP hydrolysis. A comparison of the observed ATPase activity of uncoupled labeled SMP and the activity for ATP-driven reverse electron transport in coupled labeled SMP with the corresponding values of the unlabeled control samples shows that the observed fractional inhibition ATP hydrolysis is the same for both the coupled SMP and uncoupled SMP and is determined only by the state of stereospecific labeling of F1. The effect of preincubation under simulated oxidative phosphorylation conditions on the ATPase activity of the unperturbed, specifically NBD-labeled submitochondrial particles was also examined. The data show that respiration-generated proton flux does not cause the beta subunits in bovine heart proton-ATPase to continue switching places with each other during oxidative phosphorylation. Samples of NBD-F1 with specific labels on its nonhydrolytic beta' subunits but none on its hydrolytic beta' subunit were prepared by a three-cycle process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.     

Adenosine 5'-triphosphate analogues as structural probes for Escherichia coli glutamine synthetase

Introduction of specific structural probes into substrate binding sites of Escherichia coli glutamine synthetase is now possible. Various analogues of ATP substituted with an amino or sulfhydryl moiety at the 6- or 8-position of the purine ring have been found to substitute for ATP in the autoinactivation reaction of the manganese enzyme with L-Met-(S)-sulfoximine at pH 7. Dissociation of enzyme complexes containing an ADP analogue, L-Met-(S)-sulfoximine phosphate, and 2 equiv of Mn2+ is negligible at neutral pH. Prior to binding of the mercapto nucleotides to active sites, 6-mercaptopurine ribonucleoside triphosphate (6-S-ATP) and 8-mercaptoadenosine 5'-triphosphate (8-S-ATP) also have been further modified with fluorescent and chromogenic probes for energy-transfer measurements [Maurizi, M. R., Kasprzyk, P. G., & Ginsburg, A. (1986) Biochemistry (following paper in this issue)] or with electron-dense markers for electron microscopic and X-ray crystallographic structural analyses. Binding 6-S-ATP or 8-S-ATP to enzyme active sites at pH 7.1 produced red shifts of approximately 6 nm in nucleotide spectra characteristic for transfer of these nucleotide analogues into more acidic and hydrophobic environments. The spectrum of 6-S-ADP at active sites was more red-shifted than that of 6-S-AMP attached to adenylylation sites. The thiol group at the 6- or 8-position of the purine ring of the bound nucleotides was accessible for reactions with alkylating or mercurial reagents. Alkylation or mercaptide formation produced large blue shifts in the spectrum of enzyme-bound 6-S-ADP or 8-S-ADP at active sites or of 6-S-AMP covalently bound at adenylylation sites. At least one of two tryptophanyl residues in each subunit is very near the nucleotide binding site, as evidenced by changes in tryptophanyl residue fluorescence on binding ATP, mercaptonucleotides, or other ATP analogues.