Determination of LMF Binding Site on a HSA-PPIX Complex in the Presence of Human Holo Transferrin from the Viewpoint of Drug Loading on Proteins

Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The K_a1, K_a2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×10⁵ M⁻¹, 9.7×10⁶ M⁻¹, and 2.0×10⁴ M⁻¹, 1.8×10⁵ M⁻¹, respectively, and the n₁, n₂ values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow''s site IIA to Sudlow''s site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex.     

Massive fungal contamination in animal care facilities traced to bedding supply.

During the course of immunologic studies involving the gastrointestinal colonization of mice with Candida albicans, it became apparent that the animals were being exposed to large numbers of Aspergillus fumigatus spores which interfered with the C. albicans colonization. To determine the source of the A. fumigatus exposure and the extent of fungal contamination of the medical school vivarium and four satellite facilities, fungal analyses of feed, bedding, and air were undertaken. Initial samples from the air were collected with 3-h settle plates; air sampling following cleanup was done with an Anderson air sampler. The source of contamination in the mouse rooms was determined to be Beta Chip bedding, which came from the manufacturer highly contaminated. Beta Chip bedding (1 g) obtained from the manufacturer just prior to testing contained 10(4) CFU of A. fumigatus, 20 CFU of a zygomycete, and 10 CFU of a Penicillium sp. Coarse-grade Beta Chip had approximately one-half those levels of contamination. Pure Cob bedding was highly contaminated also, but with a Fusarium sp. and a Cladosporium sp. Untreated and heat-treated Sani-Chip as well as all other heat-treated preparations obtained from the manufacturer contained no detectable spores. Rodent chow direct from the manufacturer had no A. fumigatus, although it did contain 150 CFU of fungus per g, including 80 CFU of a Rhodotorula sp., 60 CFU of Cryptococcus uniguttulatus, and 1 CFU of a Penicillium sp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbiological analysis of rock cod (Sebastes spp.) stored under elevated carbon dioxide atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO(2), 20% air) at 4 degrees C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35 degrees C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20 degrees C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20 degrees C under MA. Isolation plates incubated at 20 degrees C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Survival of human parainfluenza viruses in the South Polar environment

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

Survival of human parainfluenza viruses in the South Polar environment.

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

Increased salt- and nisin-sensitivity of pressure-injured bioluminescent Listeria monocytogenes

Suspensions of a bioluminescent (luxAB) transformant of Listeria monocytogenes in pH 7.0 phosphate buffer were pressurised and the effect of the pressure treatment was monitored by plate counting. When the bacteria were suspended in NaCl- and nisin-free buffer the number of colony forming units (CFU) decreased by 3 and 6 log cycles after 300 MPA for 10 and 30 min, respectively. Supplementing the plating medium with 5% NaCl did not influence the colony forming capacity of non-pressurised cells, however, CFU of residual populations after respective treatments of 300 MPa for 10 and 30 min were reduced by a further 2 and 3.5 log cycles in case of salt containing plates. Nisin-addition to the plating medium caused less than one log unit decrease in the CFU of the non-pressurised population. However, the CFU of 10 min-pressurised sample was 4 log cycles less in the nisin-containing plates than in the nisin-free ones, whereas no colonies were formed in the nisin-containing plates even when 1 ml was inoculated from the originally 10(10) CFU/ml population after 300 MPa for 30 min. The luciferase activities (bioluminescence intensities) decreased concomitant with the reduction of the viable cell counts, however, they were approx. 0.6-0.8 log units less in the presence of 5% NaCl in the pressurised suspension than those expected from the previously established linear correlation between the logarithmic light outputs and the logarithmic viable cell counts.     

医院病房环境真菌监测分析

目的 调查医院各病区环境中真菌含量及分布特点.方法 运用分离培养及DNA测序方法对医院不同病区、不同环境中真菌进行监测分析.结果 医院不同病区真菌含量不同,呼吸科、血液科、小儿科空气中真菌含量较高,分别为400、225和200 CFU/m3,其中以烟曲霉菌( 325 CFU/m3)、黄曲霉菌( 275 CFU/m3)、枝孢霉菌( 125 CFU/m3)、根霉( 125 CFU/m3)为主;老年病科、肿瘤科和血液科空调出气口真菌含量较高,分别为0.559、0.500和0.323 CFU/cm2,以链格孢霉(0.441 CFU/cm2)、根霉(0.412 CFU/cm2)、烟曲霉菌(0.294 CFU/cm2)为主;不同环境中真菌含量不同,其中以空气和空调出气口真菌含量最高,分别为130 CFU/m3、0.173 CFU/cm2.结论 真菌广泛存在于医院环境中,且不同病区、不同环境真菌污染程度不同,因而我们必须制订健全的消毒管理制度,预防医院真菌感染.     

Microbiological Analysis of Rock Cod (Sebastes spp.) Stored Under Elevated Carbon Dioxide Atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO₂, 20% air) at 4°C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35°C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20°C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20°C under MA. Isolation plates incubated at 20°C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.     

Role of zymogen and activated factor X as scaffolds for the inhibition of the blood coagulation factor VIIa-tissue factor complex by recombinant nematode anticoagulant protein c2

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.     

Aspergillus Spp. as an important occupational risk factor among susceptible individuals

The prevalence of different species of aspergilli was studied for two consecutive years (from April 1989 to March 1991) in a mechanised bakery, a poultry farm and a large municipal hospital in Delhi, India using an Andersen six stage volumetric sampler. A rich diversity of aspergilli (26 species) were recorded in each environment. The major contributors in each environment were Aspergillus flavus, A. niger, A. versicolor, A. sydowi, A. fumigatus, A. japonicus. The concentration of aspergilli was higher inside the work place compared to the outside air, with 25% to 45% contribution to the total fungal load. The concentration of A. flavus was higher in the storage section of the bakery and in the shed of the poultry farm, while A. niger was characteristic of the packing section and the hatchery. The hospital, which was naturally ventilated with a continuous mixing of indoor and outdoor air had a significantly high concentration of A. flavus (p < 0.05) inside the ward. Some of the frequently encountered species of Aspergillus are A. janus, A. ochraceous, A. nidulans, A. variecolor, A. luchuensis and A. terreus. Other species of Aspergillus had a very low occurrence, accounting for less than 1% of the total fungal load.     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.     

The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cellular receptor and cofactor for the activation of the plasma protease factor VII. TF activity in both monocytes and endothelial cells is regulated by various cytokines and mitogens, including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). Three TF constructs (full-length human, a cytoplasmic domain deletion mutant, and a human-rat TF chimera), expressed in a human kidney cell line, were used to examine the in vivo phosphorylation state of TF after PMA treatment. The cytoplasmic domains of both rat and human TF were rapidly phosphorylated after cells were treated with 10-100 nM PMA. This response was completely abolished by preincubating cells with staurosporine, the potent PKC inhibitor, prior to PMA treatment. Localization of the phosphorylation site(s) to the cytoplasmic domain was demonstrated using a deletion mutant of TF and by CNBr digestion at the single methionine residue (Met-210) in the TF sequence. The rat TF cytoplasmic domain was phosphorylated to a higher specific activity than the human TF cytoplasmic domain. Phosphoamino acid analysis of the chimeric TF revealed both phosphothreonine and phosphoserine, whereas human TF contained only phosphoserine. Thus both potential phosphoacceptor sites are phosphorylated in the rat TF cytoplasmic domain. Alignment of TF cDNA sequences of mouse, rat, rabbit, and man revealed that the phosphoacceptor site (X-S*/T*-P-X, where asterisk indicates the phosphorylated residue) in the cytoplasmic domain has been conserved through evolution.     

Synthesis and antifungal activity of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles

Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger (ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a-d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 microg/disc against A. fumigatus and 11.71 microg/disc against A. flavus and A. niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 microg/ml in case of A. fumigatus and 31.25 microg/ml with A. flavus and A. niger. The MIC90 value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml against A. fumigatus. The MIC90 values of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 microg/ml. The in vitro toxicity of the most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 microg/ml. The standard drug amphotericin B exhibited 100% lysis at a concentration of 37.5 microg/ml.     

Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).     

Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.     

ADP binding to TF1 and its subunits induces ultraviolet spectral changes

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.