Significance of tibial MRI findings of special forces recruits at the onset of their training.

INTRODUCTION: MRI is commonly used to evaluate medial tibial stress syndrome (MTSS), based on grading assessments developed in civilian populations. When MTSS represents stress fracture, rest is required to allow for bone remodelling to occur. False positive evaluations can lead to unnecessary recruit attrition. METHODS: Thirty randomly selected new recruits to a special forces training unit underwent MRI of their tibias using the T2-Dixon sequence at the onset of training. Evaluation was according to the Fredericson MTSS grading system. Prior to undergoing MRI, anthropomorphic measurements, a survey of sports history and an orthopaedic examination of subject tibias were performed. Orthopaedic follow-up was through 11 weeks of training. RESULTS: Medial periosteal oedema without the presence of bone marrow oedema, corresponding to a grade 1 stress reaction, was present on MRI in 10 recruits (17 tibias). In only one case did the periosteal oedema include the posterior aspect of the medial cortex where medial tibial stress fractures usually occur. Tibial tenderness was present in seven tibias on examination done just prior to the MRI studies, but none were symptomatic and only one had periosteal oedema present on MRI, but without anatomical correlation between the site of the tenderness and the periosteal oedema. During subsequent training, five tibias in four recruits developed pain and tenderness. Two had periosteal oedema in their prior MRIs, but the location did not coincide anatomically with that of the tibial tenderness. The time from stopping sports before induction and the presence of periosteal oedema was not significant. CONCLUSION: Periosteal oedema, one of the hallmarks used in MRI grading systems to evaluate MTSS, was found to have a 37.7% false positive rate for anatomically corresponding tibial tenderness at the time of the examination and during subsequent training, indicating the grading systems' low utility for the military.

Measles-related hospitalizations and associated complications in Jerusalem, 2018-2019.

OBJECTIVES: The 2018 measles outbreak in Israel affected >2000 people in Jerusalem. The aim of the study was to describe clinical features and complications of hospitalized measles patients in Jerusalem, as related to age group and risk factors. METHODS: All individuals hospitalized with measles in the three main hospitals in Jerusalem during March 2018 to February 2019 were included. Demographic, clinical and laboratory data were analysed. RESULTS: Of 161 hospitalized individuals, 86 (53.4%) were <5 years old, 16 (10%) were ≥5 years but <20 years old, and 59 (36.6%) were ≥20 years old. Most, 114/135 (85%), were unvaccinated. Immunocompromised state was identified in 12/161 (7.5%) patients, 20/161 (12.4%) had other underlying co-morbidities, and four were pregnant. Hypoxaemia on admission was a common finding in all age groups. Hepatitis was more common among adults ≥20 years old (33/59, 59%). Measles-related complications were noted in 95/161 (59%) patients, and included pneumonia/pneumonitis (67/161, 41.6%), which was more common in young (<5 years) children, diarrhoea (18/161, 11.2%), otitis (18/161, 11.2%), and neurological complications (6/161, 3.7%)-the latter occurring more frequently in the 5- to 20-year age group. Two of the 12 immunocompromised patients died of measles-related complications. A high re-admission rate (19/161, 11.8%) within 3 months was documented among hospitalized measles patients. CONCLUSION: The burden of hospitalization, as well as the high rate of short- and long-term complications observed in hospitalized patients, underscore the importance of maintaining a high measles vaccine coverage, with enhanced targeting of unvaccinated population pockets.

Erythema ab igne of shins: a kerosene stove-induced prototype in diabetics.

A patient with erythema ab igne of shins is presented, caused by repeated thermal injury induced by a heating stove placed between the knees. This injury pattern has been repeatedly identified in diabetic patients involved in similar heating practice, underscoring a possible predisposition related to diabetic neuropathy.

The effect of high versus low loading on bone strength in middle life.

While bone mass and geometry are largely genetically determined, mechanical loading is considered to be an important additional determinant. This study investigates to what extent very high mechanical loading begun at a young age and sustained afterward can affect tibia bone mass and geometry in middle age. Cohorts from a common ethnic background, with a history of very high and very low tibia bone loading based on an assessment of their activities according their strain levels were compared. The study hypothesis was that the tibia bone density and geometric strength parameters would be greater in the high bone loading cohort. Subjects from a group of elite infantry recruits who sustained a 31% incidence of stress fractures during their basic training in 1983, were reviewed 25 years later. The tibia bone strength of 25 of these soldiers, 11 of whom had sustained stress fractures, was compared to a group of 20 subjects who received exemption from military service in 1982-5 because they were religious scholars and who continued these studies afterwards. Anthropometric measurements were made. The bone density and geometric strength of the tibia was assessed by quantitative computerized tomography (QCT). The average daily dietary intake and metabolic expenditure of subjects were assessed by questionnaires. At the 25 year follow-up soldiers were on an average 3 cm taller than the religious scholars (p=0.02) and had lower abdominal girths (p=0.03). There was no difference in the tibia cortical density between cohorts in spite of the fact that the religious scholars had lower daily calcium intakes (p=0.02). Soldiers had stronger tibias based on geometric engineering criteria. The mean area moments of inertia (p=0.02, p=0.04) and polar moments of inertia (p=0.02) were 16% larger in the soldier cohort. By multivariate regression analysis greater height, weight and daily energy expenditure were related to larger bone geometric strength parameters. According to semipartial eta-square analysis, between 39% to 45% of the variance in the area moments of inertia between the cohorts was attributable to these three parameters. The religious scholars burned less calories daily, principally because they did no sport activity (p=0.001). There was no difference in tibia bone strength parameters between soldiers who did and did not sustain stress fractures in their 1983 basic training. In conclusion, in a middle age population with a common ethnic origin, the high bone loading cohort had stronger tibias than the low bone loading cohort based on larger geometric strength properties and not because of higher cortical density. In spite of being at the extremes of the bone loading spectra, the tibia area moment of inertia of the two cohorts in this study differed by only 16%, with part of this difference attributable to factors other than bone loading. We do not know for sure if the difference in the geometric properties is related to high bone loading or whether people with stronger bones are more likely to engage in high bone loading. Healthy male subjects who sustained stress fractures at a young age do not have weaker tibias at middle age according to QCT measurements.

Characteristics of protection by MgADP and MgATP of α3ß3γ subcomplex of thermophilic Bacillus PS3 ßY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis.

MgADP and MgATP binding to catalytic sites of ßY341W-α(3)ß(3)γ subcomplex of F(1)-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 µM and 12 mM, and 46 µM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K(d) of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with ßY341W-α(3)ß(3)γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F(1)-ATPases.

Probes of inhibition of Escherichia coli F(1)-ATPase by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in the presence of MgADP and MgATP support a bi-site mechanism of ATP hydrolysis by the enzyme.

Binding of MgADP and MgATP to Escherichia coli F(1)-ATPase (EcF(1)) has been assessed by their effects on extent of the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). MgADP at low concentrations (K(d) 1.3 microM) promotes the inhibition, whereas at higher concentrations (K(d) 0.7 mM) EcF(1) is protected from inhibition. The mutant betaY331W-EcF(1) requires much higher MgADP, K(d) of about 10 mM, for protection. Such MgADP binding was not revealed by fluorescence quenching measurements. MgATP partially protects EcF(1) from inactivation by NBD-Cl, but the enzyme remains sensitive to NBD-Cl in the presence of MgATP at concentrations as high as 10 mM. The activating anion selenite in the absence of MgATP partially protects EcF(1) from inhibition by NBD-Cl. A complete protection of EcF(1) from inhibition by NBD-Cl has been observed in the presence of both MgATP and selenite. The results support a bi-site catalytic mechanism for MgATP hydrolysis by F(1)-ATPases and suggest that stimulation of the enzyme activity by activating anions is due to the anion binding to a catalytic site that remains unoccupied at saturating substrate concentration.

Metabolism of gonadotropins: comparisons of the primary structures of the human pituitary and urinary LH beta cores and the chimpanzee CG beta core demonstrate universality of core production.

The gonadotropins are a family of closely related heterodimeric glycoprotein hormones homologous in structure to disulfide-knot growth factors. Metabolic proteolytic processing in vivo of this disulfide cross-linked region results in urinary excretion of a residual highly stable core structure. The primary structure of the pituitary form of the hLH beta core was reported earlier, but it has proved difficult to isolate the urinary core, although antibodies to the pituitary core demonstrated its presence. By conventional and immunoaffinity methods, the urinary core has been isolated and its structure determined by both chemical and mass spectrometric methods. The urinary hLH beta core is the same as the pituitary-extracted hLH beta core, beta 6-40 disulfide bridged to beta 55-93, except that the pituitary core is more heterogeneous containing also beta 49-93. These findings imply a dual origin of urinary cores, both directly from a secreting tissue and by kidney processing of circulating hormone. We also found that pregnant chimpanzees excrete a CG beta core with a primary structure identical to that of the human CG beta core of pregnancy. In conclusion, gonadotropin core generation and urinary excretion of nearly identical gonadotropin metabolites is common among primates. Although possible biological functions of these core fragments remain unproven, they have diagnostic utility because of their stability and abundance.

Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.

Nucleotide-depleted beef heart F1-ATPase exhibits strong positive catalytic cooperativity.

Catalytic cooperativity is a central feature of the binding change mechanism for F0F1-ATP synthases. However, in a recent publication (Reynafarje, B. D., and Pedersen, P. L. (1996) J. Biol. Chem. 271, 32546-32550), Reynafarje and Pedersen claim that cooperative effects are an artifact caused by endogenous nucleotides and that when such nucleotides are removed, the multiple catalytic sites on MF1 behave independently during ATP hydrolysis. In contrast to this conclusion, we show here that when ATP is loaded at a single catalytic site on nucleotide-depleted MF1, the rate of product release is accelerated by up to 5 x 10(4)-fold by the binding of ATP at adjacent catalytic sites. Hence, nucleotide-depleted MF1 is not an exception but does in fact show strong cooperative interactions. In addition, evidence is presented supporting a random order for product release during ATP hydrolysis.

Studies on reconstitution of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase.

The energy-transducing nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum is composed of 3 subunits alpha 1, alpha 2 and beta, with M(r) values, respectively, of 40.3, 14.9 and 47.8 kDa. Subunit alpha 1 is water-soluble, loosely bound to chromatophores, and can be easily and reversibly removed. Subunits alpha 2 and beta are integral membrane proteins, and their removal from chromatophores requires the use of detergents. Treatment of chromatophores with various detergents inhibited reconstitution of transhydrogenase activity when alpha 1 was added to the detergent-treated chromatophores. This apparent inhibition could be reversed by addition of a divalent metal ion. The best condition for extraction of alpha 2/beta from chromatophores was the use of 1% deoxycholate in the presence of 0.34 M KCl. Under these conditions, the extracted alpha 2/beta mixed with purified alpha 1 was completely inactive, but gained full activity when the assay medium was supplemented with 2-3 mM MgCl2 or CaCl2. It was shown that metal ions had little effect on the apparent Km of substrates, but greatly increased the affinity between purified alpha 1 and the detergent-treated or detergent-solubilized alpha 2/beta. It seems possible that the R. rubrum transhydrogenase contains a detergent-extractable metal ion, which is required for proper binding of the soluble alpha 1 subunit to the chromatophore-bound alpha 2/beta subunits.

Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi. No binding of GDP at non-catalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity. However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone. For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used. With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase. Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants.

Structure of cucumarioside G2, a novel nonholostane glycoside from the sea cucumber Eupentacta fraudatrix.

The new triterpene glycoside cucumarioside G2 [1] has been isolated from the sea cucumber Eupentacta fraudatrix. Glycoside 1 is the first triterpene glycoside with the 23,24,25,26,27-pentanorlanostane type of aglycone. Its structure has been established by chemical transformations as well as 13C- and 1H-nmr, eims, and liquid sims studies.

Nucleotide binding sites on beef heart mitochondrial F1-ATPase. Cooperative interactions between sites and specificity of noncatalytic sites.

We have studied the properties of beef heart mitochondrial F1 having inhibitory MgADP bound at one of the three catalytic sites and various levels of occupancy of the three noncatalytic nucleotide sites including zero, two, or three ADP/ATPs or two ADP/ATP plus one GTP. The properties examined include the rate of MgATP-dependent reactivation and the rate of increase in the fraction of F1 containing transiently bound intermediates. For each form of the enzyme tested, the rate of reactivation closely paralleled the rate of increase in the level of bound intermediates, indicating that when one catalytic site on F1 is blocked by inhibitory MgADP, the remaining two sites are incapable of residual uni- or bi-site activity. It was also found that the stability of the MgADP-inhibited complex decreases with full occupancy of the noncatalytic sites. This demonstrates that the noncatalytic sites modulate the properties of catalytic sites. Finally, it was found that the noncatalytic sites on mitochondrial F1 do not, as has long been believed, bind adenine nucleotides exclusively. Evidence is presented that both GTP and PPi bind tightly at noncatalytic sites.

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.

When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites.

Inactivation of the isolated ATPase portion of ATP synthase from beef-heart mitochondria (F1) by its natural inhibitor protein (IP) during steady-state ATP hydrolysis is accompanied by a trapping of 1 mol nucleotide/mol F1 in one of the catalytic sites. The trapped nucleotide is not released during incubation of IP-inhibited F1 in the presence of MgATP at pH 8.0 for at least 20 min, indicating a very low turnover rate of the IP.F1 complex. The ATP/ADP ratio of the trapped nucleotides is higher than that found for transitorily bound nucleotides under the same conditions but in the absence of IP. The IP impairs the acceleration of ATP hydrolysis and product release steps that results from the binding of ATP to an alternate catalytic site. It also inhibits ATP hydrolysis by a single catalytic site or shifts the equilibrium toward ATP formation from bound ADP and Pi. At high pH, an active acidic form of the free IP is transformed to the inactive basic one with a half-time of 3-4 s. This process seems to be prevented by IP binding to F1. The inactive basic form of IP does not compete with the active acidic IP for the binding to F1. The data do not favor the existence of a long-lived catalytically active IP.F1 intermediate during IP action on F1. The reactivation of IP-inhibited membrane-bound F1 by energization may be due to a conformational change in the IP.F1 complex allowing the transformation of IP into an inactive basic state that rapidly dissociates.

Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.

The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

The ADP that binds tightly to nucleotide-depleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site.

Previous studies have shown that the initial complex formed when ADP binds to nucleotide-depleted F1-ATPase is transformed with a half time of 2 to 3 min to form with a much lower rate of ADP release. The ADP binding results in a strong inhibition of ATPase activity. The present paper reports appraisal of where the inhibitory ADP binds by use of the photoreactive ADP analog, 2-N3-ADP. In presence of Mg2+ the 2-N3-ADP like ADP induces reversible inhibition of nucleotide-depleted F1 (ndF1) with a Kd of about 10 nM. Photoirradiation of the inactive 2-N3-[beta-32P]ADP-ndF1 complex results in labeling of only the beta-subunit. The major labeled peptide isolated from a trypic digest consists of residues from Ala-338 to Arg-356, with Tyr-345 as the site of labeling. This identifies the site of the inhibitory ADP binding as one of the catalytic sites of the enzyme.

The F1-type ATPase in anaerobic Lactobacillus casei.

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.