Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity

Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These measurements indicate that, over this NaCl concentration range, hammerhead ribozyme activity is influenced by population of a specific set of divalent cation sites.     

Characterisation of L-[3H]glutamate binding to fresh and frozen crude plasma membranes isolated from cerebral cortex of adult rats

Specific [3H]glutamate binding to fresh crude plasma membranes (CPMs) was compared with binding to frozen CPMs and the optimal conditions for the binding to frozen CPMs isolated from cerebral cortex of adult rats were determined. Freezing reduced [3H]glutamate binding (3.5-fold), and pre-incubation of previously frozen membranes followed by three washes increased binding (4.5-fold) when compared to fresh samples. CPMs washed once, pre-incubated at 37 degrees C and washed 3 times was adopted as the most adequate condition for the binding assay of frozen membranes. In a Cl(-)-containing medium, [3H]glutamate binding (Bmax=97.9 pmol/mg, Kd=349.68 nM) to this frozen CPM preparation was significantly displaced by excess quisqualic acid (QA) (65%), L-2-amino-4-phosphonobutyric acid (L-AP4) (35%), trans-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) (25%) and alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (25%). In a Cl(-)-free medium, binding (Bmax=44.14 pmol/mg, 311 nM) was significantly displaced by QA (45%), L-AP4 (25%), ACPD (25%), AMPA (25%), kainic acid (20%) and N-methyl-D-aspartate (15%).     

Role for anions in pulmonary endothelial permeability

beta-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl- flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl-, Br-, I-, F-, acetate (Ac-), gluconate (G-), and propionate (Pr-). Permeability to albumin (Palbumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl-, the Palbumin was 3.05 +/- 0.86 x 10(-6) cm/s and fell by 70% with the addition of IBMX. The initial Palbumin was lowest for Pr- and Ac-. Initial Palbumin was higher in Br-, I-, G-, and F- than in Cl-. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl- was the extracellular anion, and the order among halide anions was Cl- > Br- > I- > F-. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]o did not systematically affect endothelial permeability in this system. When Cl- was the extracellular anion, varying [Ca2+]o from 0.2 to 2.8 mM caused a change in initial Palbumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4'-isothiocyanotostilbene-2, 2'-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial Palbumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.     

Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5'-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.     

Effects of anions on ATP-activated nonselective cation current in NG108-15 cells

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl- < I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.     

Thermoluminescence of BaFCl_xBr_（1－x）：Sm System and Assignment of Their Glow Peaks

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Smoking and female infertility: a systematic review and meta-analysis

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.     

Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells

This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37 degrees C allowed the calculation of a kinetic Kd of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37 degrees C for 90 min indicated the presence of a single high-affinity site (Kd = 3.87 +/- 0.60 nM), saturable and abundant (Bmax = 9.64 +/- 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1-7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-(N alpha -benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 microM. This binding site is, therefore, distinct from angiotensin II receptors, AT1 and AT2. Addition of the divalent cations Mg2+, Mn2+ or Ca2+ to the incubation buffer resulted in 90-95% inhibition of the [125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites (Bmax = 17.8 +/- 2.5 pmol/mg protein), with no change in affinity (Kd = 5.7 +/- 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTP gamma S, had no effect on [125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 +/- 2.0 nM; n1 = 170 +/- 32 sites per cell vs. Kd1 = 7.2 +/- 1.1 nM; n1 = 23 +/- 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 +/- 0.1 microM; n2 = 1,832 +/- 232 sites per cell vs. Kd2 = 1. 6 +/- 0.1 microM; n2 = 1,740 +/- 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Relationship of variations in tumor cell kinetics induced by primary chemotherapy to tumor regression and prognosis in locally advanced breast cancer

We have studied binding of isradipine to A7r5 vascular smooth muscle cells as a function of membrane potential and cell proliferation. Consistent with a voltage-modulated receptor model, two classes of binding sites were detected in confluent cultures: high-affinity sites under depolarizing (50 mM K+) conditions (Kd = 45 +/- 3 pM), and lower affinity sites under resting (5 mM K+) conditions (Kd = 181 +/- 20 pM). However, proliferating cells also displayed the high-affinity state at rest (Kd = 29 +/- 9 pM) in addition to a low-affinity site (Kd = 869 +/- 383 pM). Analysis of dissociation rates also revealed two receptor classes during proliferation. Proliferating cells showed a single class of high-affinity sites (Kd = 39 +/- 6 pM) when depolarized, similar to confluent cells. Receptor density in confluent monolayers increased from 15 +/- 3 fmol/10(6) cells at 5 days to 72 +/- 6 fmol/10(6) cells after 10 days. These results suggest (i) that some L-type Ca2+ channels are spontaneously active in proliferating vascular smooth muscle cells, but require depolarization to activate in a confluent monolayer, and (ii) that the density of dihydropyridine receptors increases after a monolayer becomes confluent.     

Comparison of binding parameters of sigma 1 and sigma 2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes

Comparisons of binding parameters of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) at sigma binding sites in guinea pig and rat brain membranes demonstrated that [3H](+)-pentazocine binds to a single high-affinity site, whereas [3H]DTG binds to two high-affinity sites in both species. The Kd values of the radioligands were similar in both types of membranes. However, the density of sigma 1 sites in guinea pig was significantly higher than that of rat. Novel trishomocubanes were tested for their affinities at sigma 1 and sigma 2 binding sites in guinea pig brain membranes using [3H](+)-pentazocine and [3H]DTG as the radioligands. N-(4-Phenylbutyl)-3-hydroxy-4- azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane (ANSTO-14) showed the highest affinity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity, as a result of increasing the alkyl chain between the cubane moiety and the aromatic ring. N-(3'-Fluorophenyl)methyl- 3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11]dodeca ne (ANSTO-19), displayed the highest affinity for sigma 2 sites (Ki = 19.6 nM) and 8-fold sigma 2/sigma 1 selectivity due to a fluoro substitution in the meta position of the aromatic ring. These represent structurally novel lead compounds, especially for the development of selective sigma 2 receptor ligands.     

Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead

We have characterized the specific binding of glucagon in hepatocytes isolated from two teleost species, the American eel (Anguilla rostrata) and the brown bullhead (Ictalurus nebulosus). Specific glucagon binding was 9.3 and 10.7% in bullhead and eel hepatocytes respectively, after a 2-h incubation at 12 degrees C. Curvilinear Scatchard plots suggest the presence of two classes of binding sites with apparent dissociation constants (Kd) of 1.97 nM (high affinity) and 17.3 nM (low affinity) for bullhead and 2.68 and 22.9 nM for eel cells. The number of high-affinity binding sites per cell was significantly higher in the eel (10,413) than in the bullhead (3811). The number of high-affinity insulin-binding sites was approximately two times higher than that for glucagon in bullheads and the opposite in the eel hepatocytes. In competition experiments, insulin did not displace 125I-labelled glucagon binding in the hepatocytes of either species, while glucagon-like peptide-1(7-37) (GLP-1) displaced glucagon but only at high concentrations, suggesting separate glucagon- and GLP-1-binding sites. The rate of dissociation of hepatocyte-bound 125I-labelled glucagon was similar for both species. Preincubation of hepatocytes in 100 nM glucagon decreased the number of high-affinity glucagon-binding sites by approximately 55% in both species, while the Kd values remained unchanged. Glucagon bound to the cell surface is internalized by fish hepatocytes. These properties indicate that the glucagon binding to hepatocytes of these two teleost species is similar to that reported for mammalian hepatocytes.     

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.     

Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam > Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.     

Genetics of idiopathic scoliosis

To define the luminal agent(s) responsible for the reduction of nephron filtration rate following increases of loop of Henle flow rate early proximal flow rate (EPFR) during loop perfusion with 17 different salt solutions were compared to the non-perfused tubules. During orthograde microperfusions a reduction of EPFR as indication of a feedback response was noted with a number of monovalent Cl- and Br- salts (LiCl, KCl, NaCl, RbCl, CsCl, NH4Cl, choline Cl, NaBr, KBr), with Na+ salts except Na acetate (NaHCO3, NaNO3, NaF, NaI, NaSCN), and with CaCl2 and MgCl2. These latter 2 solutions where used in a concentration of 70 mM while all other solutions had a concentration of 140 mM. During retrograde perfusion from the distal to the proximal end of the loop of Henle EPFR fell significantly with Cl- and Br- salts with percentage changes of EPFR ranging from -8.0 to -44.3%. In contrast, Cl- free salts and Cl- salts of divalent cations were associated with percentage changes of EPFR ranging from +7.1 to -6.2%, significance being reached only during perfusion with NaSCN. When furosemide (5 x 10(-4) M) was added to NaBr or KBr a feedback response was not observed. During orthograde perfusion with NaNO3 distal Cl- concentrations were 44.2 +/- 5.08, mM (mean +/- S.E.) at a perfusion rate of 10 nl/min and 59.1 +/- 3.93 mM at a rate of 40 nl/min. CaCl2 perfusion induced a marked elevation of distal Cl- concentrations to levels higher than 140 mM. Loop chloride handling was normal during RbCl perfusion. The magnitude of the feedback response during retrograde perfusion was not changed by lowering NaCl concentration from 140 to 60 mM, but fell when NaCl concentration was further reduced. In contrast to orthograde perfusions it was insensitive to changes in flow rate. Our results are compatible with the thesis that feedback responses depend critically upon the rate of Cl- transport probably across the macula densa cells. Br- ions can replace Cl- because they appear to share a common transport pathway which can be inhibited with furosemide. Unspecificity of feedback responses during orthograde microperfusions is due to presence of Cl- ions in the macula densa region even when solutions are initially Cl- free. Cl- salts of divalent cations do not elicit a feedback response because Cl- transport is severely curtailed.     

Hydrogen atoms are produced when tryptophan within a protein is irradiated with ultraviolet light

The UV photolysis of the aromatic amino acid, tryptophan (Trp), in the Ca(2+)-binding protein, cod parvalbumin, type III, was studied using electron paramagnetic resonance (EPR) spectroscopy in the temperature range 4-80 K. For the Ca(2+)-bound protein, irradiation with UV light (250-400 nm) resulted in the generation of atomic hydrogen with a hyperfine splitting of 50.9 mT, whereas in the Ca(2+)-free form, where the Trp is exposed to solvent, the trapped atomic hydrogen was not in evidence. In the same spectra, the radical signal in the g = 2.00 region could be detected. The line shape of the Ca(2+)-bound form is similar to the EPR line shape obtained for Trp in micellar systems. In contrast, the EPR line shape for the Ca(2+)-free form is essentially featureless up to 80 K. The EPR spectra of the photoproducts of Trp and the nature of the photoreactions are therefore sensitive to the environment of Trp within the protein.     

Interaction of manganese with bovine prothrombin and its thrombin-mediated cleavage products

The binding of the paramagnetic metal, Mn(II), to bovine prothrombin and the thrombin-mediated cleavage products of prothrombin, i.e. fragment 1 and the prethrombin 1 has been investigated. Analysis of the Scatchard plots of the binding data reveals that prothrombin has two high affinity Mn(II) binding sites with a Kd of 1.2 +/- 1.0 X 10(-5) M and approximately two to three lower affinity Mn(II) sites with a Kd of 1.3 +/- 1.0 X 10(-4) M. Positive cooperativity in Mn(II) binding to prothrombin was observed for the strong sites. Fragment 1, the phospholipid-binding region of prothrombin, possesses two high affinity Mn(II) sites with a Kd of 2.2 +/- 1.0 X 10(-5) M and at least two lower affinity sites with a Kd of approximately 2.5 +/- 1.0 X 10(-4) M. Positive cooperativity was not observed for the binding of Mn(II) to fragment 1. Prethrombin 1 binds one Mn(II) with a Kd of 3.2 +/- 1.0 X 10(-4) M. Using the values of free Mn(II) concentration, as determined by EPR measurements and the observed enhancements of the water proton relaxation rates at various concentrations of Mn(II) and protein, the binary enhancement values (epsilon b) of the metal-protein complexes were obtained. The extrapolated values are 11 +/- 0.4 for the initial prothrombin-binding sites, and 10 +/- 0.3 for the tight binding sites of fragment 1. The unique epsilon b value obtained for prethrombin 1 was 5.3 +/- 0.7. When Mn(II) was used in a Factor Xa-metal ion-phospholipid system for activation of prothrombin, the rate of generation of thrombin was less than or equal to 5% of that obtained when Ca(II) was employed in this activation system. Addition of Mn(II) to the same activation system containing Ca(II) resulted in a marked decrease in the rate of thrombin generation, suggesting that Mn(II) probably competes for the same sites on prothrombin as Ca(II). In agreement with this is the observation that the Mn(II) sites on prothrombin could be displaced by Ca(II) at high concentrations of Ca(II).     

Cortical spreading depression protects against subsequent focal cerebral ischemia in rats

Specific, high-affinity angiotensin II (A II) receptors were observed on granulosa and thecal cells of preovulatory ovarian follicles from immature PMSG-treated rabbits. Scatchard analysis of 125I-[Sar1,Ile8]A II binding to freshly prepared cells was indicative of only one class of binding sites. Kd values were 0.26 +/- 0.11 nM and 0.18 +/- 0.02 nM, densities of A II receptors were 0.06 +/- 0.02 fmol/10(5) cells and 0.08 +/- 0.01 fmol/10(5) cells for granulosa and thecal cells, respectively. When cells were incubated for 48 h with hCG, Kd values were of the same order of magnitude, but the amount of A II receptors was increased 2-fold in granulosa and 4-fold in theca. Using subtype specific ligands (Losartan for AT1 and PD 123319 for AT2) in competitive binding experiments, A II receptors were found to be of the AT1 type on both granulosa and thecal cells freshly prepared or incubated 48 h in vitro. These results establishing the existence of high affinity AT1 receptors on the two cell types of the rabbit preovulatory follicles contrast with previous observations showing the presence of AT2 receptors on granulosa or theca from several species.