Arrangement and number of clustered regularly interspaced short palindromic repeat spacers are associated with erythromycin susceptibility in emm12, emm75 and emm92 of group A streptococcus.

Clustered regularly interspaced short palindromic repeats (CRISPR) are composed of numerous repeat-spacer units and are considered a prokaryotic defence system against foreign nucleic acids. Since antibiotic-resistant genes are frequently encoded in foreign nucleic acids, the aim of this study was to test whether erythromycin susceptibility in group A streptococcus (Streptococcus pyogenes) is associated with characteristics of CRISPR elements. Erythromycin susceptibility of 330 isolates collected between 1997 and 2003 was analysed. Among 29 emm types, emm12, emm75 and emm92 showed significant changes in erythromycin-resistance rates. By sequencing the spacers from two CRISPR loci, spacer contents in emm12, emm75 and emm92 strains were associated with erythromycin susceptibility. Strains with fewer spacers were more resistant to erythromycin. Moreover, in emm4 strains, which showed no significant change in their annual erythromycin-resistance rate, CRISPR type and number of spacers were not correlated with erythromycin susceptibility. These results highlight a novel association between CRISPR spacer content and erythromycin susceptibility in group A streptococcus.

Improvements in endotoxemic syndromes using a disintegrin, rhodostomin, through integrin αvß3-dependent pathway.

BACKGROUND AND OBJECTIVES: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg-Gly-Asp-containing snake venom disintegrin, on lipopolysaccharide (LPS)-activated phagocytes in vitro and LPS-induced endotoxemia in vivo. METHODS AND RESULTS: Rn inhibited adhesion, migration, cytokine production and mitogen-activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti-αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP-1, Rn bound to LPS-activated THP-1 and specifically blocked anti-αvß3 mAb binding to THP-1. Binding assays proved that integrin αvß3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS-induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS-induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor-α (TNF-a), interleukin (IL)-6, -1ß and -10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS-induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. CONCLUSIONS: Rn may interact with αvß3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS-induced endotoxemia may be attributed to its anti-inflammation activities in vivo.

Group A streptococcal infection caused by emm1 strains among children in southern Taiwan.

The aim of this study was to characterize the molecular epidemiology of invasive and non-invasive group A streptococcus (GAS) infections in children from 1997 through 2004 in southern Taiwan. A collection of 32 invasive and 150 non-invasive isolates were recruited for analysis. emm1 (34.4%) and emm12 (40.0%) predominated in the invasive and non-invasive isolates, respectively. The peak incidence of invasive GAS infection (IGASI) occurred between 2002 and 2003. emm4 and emm12 were the major types among clinical isolates before 2001, and was replaced by emm1 during 2002-2003. All emm1 isolates were clonal relatedness. The declined prevalence of erythromycin resistance occurred in the major shift of the endemic isolates to emm1 strains during 2002-2003 in the community.

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

The catalase activity of Nalpha-acetyl-microperoxidase-8.

Nalpha-Acetylated microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for peroxidases. We report here that Ac-MP-8 catalyzes catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The catalase activity of Ac-MP-8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The Km and kcat of the decomposition of hydrogen peroxide (H2O2) catalyzed by Ac-MP-8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (kcat/Km) of Ac-MP-8 in catalase-type reaction of H2O2 is 100.2,/m/s, which is 5- to 12- and 50- to 100-fold less than those of MPs in cytochrome P450-type reaction of aniline/H2O2 and peroxidase-type reaction of o-methoxyphenol/H2O2, respectively. These results indicate that Ac-MP-8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroxidase-type > cytochrome P450-type > catalase-type reactions. Comparisons of the enzyme activities of Ac-MP-8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.

Evidence for the existence of some dissociation in an otherwise strong linkage disequilibrium between mitochondrial and chloroplastic genomes in Cyclobalanopsis glauca.

Variations in mitochondrial DNA in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. were studied in 140 trees from 32 populations collected from within the tree's natural range. By sequencing two mitochondrial DNA intron fragments (nad4/3-nad4/4r and nad7/2-nad7/3r), we revealed a total of 1788 bp and five polymorphic sites which allowed us to distinguish six mitotypes. The mitochondrial DNA markers provided replicated data to support population phylogeographical scenarios suggested previously using chloroplastic DNA markers. The gene genealogical tree of mitochondrial DNA was partially congruent with the chloroplastic DNA tree owing to the slower mutation rate and different mutational direction. Significant linkage disequilibrium existed between the two organellar genomes. Further paring analyses between fragments synthesized using different primers, accompanied by exclusion of polymorphic sites, showed that the random association could be attributed specifically to one of the polymorphic sites of the petG-trnP fragment of the chloroplastic genome, and the three polymorphic sites of the nad4/3-nad4/4r fragment of the mitochondrial genome. The former was inferred to derive from paternal leakage, and the latter from recurrent mutation. These polymorphic sites were also responsible for uncoupling of the combined gene tree of mitotype and chlorotype. In conclusion, specific fragments found in this study contribute to the incomplete congruence of the two organellar lineages that otherwise associate well phylogeographically.

Cordyceps sinensis and its fractions stimulate MA-10 mouse Leydig tumor cell steroidogenesis.

The effects of Cordyceps sinensis (CS) and its extracted fractions on steroidogenesis in MA-10 cells were determined. Different concentrations of CS and 3 fractions of CS (F1, a water-soluble polysaccharide; F2, a water-soluble protein; and F3, a poorly water-soluble polysaccharide and protein) were added to MA-10 mouse Leydig tumor cells with or without human chorionic gonadotropin (hCG), and the production of steroid and the expression of steroidogenic acute regulatory protein (StAR) were examined. The results showed that CS alone (2-10 mg/mL) stimulated MA-10 cell progesterone production in a dose-dependent relationship. Fractions F1 and F3 (2-10 mg/mL) also had significant (P < .05) stimulatory effects on MA-10 cell steroidogenesis with a dose-dependent relationship. However, fraction F2 did not have an effect on MA-10 cells. CS and F3, but not F1, significantly induced more steroid production in hCG-stimulated MA-10 cells (P < .05). As a temporal relationship, F1 and F3 (2 mg/mL) maximally stimulated progesterone production between 1 and 3 hours after stimulation in MA-10 cells. In addition, CS and F3 significantly enhanced MA-10 cell StAR protein expression, which indicates that CS and F3 may use a cyclic adenosine monophosphate signal transduction pathway to activate MA-10 Leydig cell steroidogenesis in a manner to that of luteinizing hormone.

Role of alpha(3)beta(1) integrin in tubulogenesis of Madin-Darby canine kidney cells.

BACKGROUND: We isolated several Madin-Darby canine kidney (MDCK) subclones that exhibit different degrees of branching tubulogenesis in lower concentrations of collagen gel. The M634 clone formed cell aggregates in 0.3% collagen gel, but developed branching tubules vigorously in 0.1% collagen gel. In contrast, the Y224 clone formed cysts in 0.3% collagen gel and displayed fewer branching structures in 0.1% collagen gel. Morphologically, M634 cells exhibited higher levels of cell scattering as well as collagen-induced cell migration than Y224. We conducted this study to delineate the underlying mechanism of branching tubulogenesis in M634 cells. METHODS: Components of the focal contact machinery were analyzed in both cell lines, including the extracellular matrix glycoproteins fibronectin, laminin, and vitronectin; cytoskeleton-associated elements alpha-actinin, talin, and vinculin; and receptors for extracellular matrix and alpha(2), alpha(3), alpha(5), alpha(v), beta(1), and beta(3) integrins. Furthermore, we established several stable transfectants of alpha(3) integrin antisense RNA in M634 cells to examine the role of alpha(3)beta(1) integrin in branching morphogenesis directly. RESULTS: There were no obvious differences in levels of the focal adhesion complex proteins between M634 and Y224 cells, except that the content of the alpha(3) and beta1 integrins were 1.2- and 0.6-fold higher in M634 cells, respectively. The expression of alpha(3) integrin antisense RNA significantly lowered the levels of alpha(3) integrin mRNA and protein. The potential of cell scattering, migration, and branching tubulogenesis in M634 cells was inhibited according to the decrease in alpha(3) integrin expression. CONCLUSION: Our data indicate that expression of alpha(3)beta(1) integrin regulates cell scattering, migration, and branching tubulogenesis of MDCK cells, possibly via adhesion to or serving as a signaling molecule for type I collagen.

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation.

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.

Role of fibronectin deposition in branching morphogenesis of Madin-Darby canine kidney cells.

BACKGROUND: Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of hepatocyte growth factor (HGF) form branching tubules. The tubule-lining epithelial cells are polarized with the basolateral surface in contact with the collagen gel and the apical surface facing the lumen. To delineate whether MDCK branching tubules construct the basal lamina, we characterized the composition of the extracellular matrix deposited by MDCK tubules. The tubule-lining cells produced an apparently incomplete basal lamina containing a discontinuous laminin substratum. In addition, a thick layer of fibronectin surrounded the basal cell surface of the branching tubule. In an attempt to delineate the role of fibronectin deposition in branching morphogenesis, we conducted this study. METHODS: MDCK cells cultured in collagen gel were employed. We first used arginine-glycine-aspartate peptides containing disintegrin rhodostomin to disturb the interactions between fibronectin and cell surface integrins. Furthermore, we established several stable transfectants expressing fibronectin antisense RNA to examine the role of fibronectin in branching morphogenesis directly. RESULTS: Rhodostomin inhibited the formation of branching tubules. The transfectants expressing fibronectin antisense RNA exhibited relatively lower levels of synthesized fibronectin and markedly diminished growth rates of branching tubules than the control clone. An inhibition of branching morphogenesis induced by the overexpression of fibronectin antisense RNA was manifested by the decrease in cell growth rates and cell migration. CONCLUSION: These results indicate that the deposition of fibronectin underlying the tubule-lining epithelium serves to enhance cell proliferation and migration, and hence facilitates the branching tubulogenesis of MDCK cells.

Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells.

BACKGROUND: Madin-Darby canine kidney (MDCK) cells cultured within collagen I gel exhibit clonal growth and form spherical multicellular cysts. The cyst-lining epithelial cells are polarized with the basolateral surface in contact with the collagen gel and the apical surface facing the lumen. To understand whether MDCK cysts construct the basal lamina, we characterized the composition of the extracellular matrix deposited by MDCK cysts. The cyst-lining cells produced an apparently incomplete basal lamina containing a discontinuous laminin substratum. In addition, the basal cell surface of the cyst was surrounded by a thick layer of fibronectin. This study was conducted to delineate the role of fibronectin deposition in cystogenesis. METHODS: MDCK cells cultured in collagen gel were employed. We first used Arg-Gly-Asp (RGD) peptides containing disintegrin rhodostomin to disturb the interaction between fibronectin and the cell surface integrin. We then established several stable transfectants expressing the fibronectin antisense RNA and with which to directly examine the role of fibronectin in cystogenesis. RESULTS: Rhodostomin markedly decreased the growth rates of the MDCK cyst, suggesting the importance of a normal interaction between fibronectin and integrins. The stable transfectants overexpressing the fibronectin antisense RNA exhibited relatively lower levels of fibronectin and markedly lower cyst growth rates than the control clone. The lower growth rate was correlated with an increase in collagen gel-induced apoptosis. CONCLUSIONS: The results indicate that the deposition of fibronectin underlying the cyst-lining epithelium serves to prevent apoptosis induced by three-dimensional collagen gel cultures, and hence facilitates cyst growth of MDCK cells.

Perturbation of platelet adhesion to endothelial cells by plasminogen activation in vitro.

To investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as epsilon-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Interaction of streptokinase and plasminogen. Studied with truncated streptokinase peptides.

The interaction of streptokinase (SK) with human plasminogen (HPlg) was investigated using truncated SK peptides prepared by gene cloning techniques. SK(16-414) and SK(16-378) could activate HPlg as efficiently as the authentic SK. SK(60-414), which had been preincubated with SK(1-59), could also activate HPlg. SK(91-414), SK(127-414), and SK(158-414), at a concentration of one-tenth of HPlg, all failed to activate HPlg. However, the truncated SK peptides in complexes with equimolar HPlg could form amidolytically active virgin enzymes that slowly converted to human plasmin (HPlm) after a lag period of 15 min. SK(16-316) could not activate HPlg. No virgin enzyme was detected when SK(16-316) was incubated with equimolar HPlg, but the HPlg in the complex was modified to HPlm after reaction for 20 min. SK(220-414) and SK(16-251) had no ability to transform HPlg to virgin enzyme or to HPlm in equimolar complex with HPlg, although they could bind to HPlg. The functions of five regions in the SK molecule (a, Ile1-Lys59; b, Ser60-Asn90; c, Val158-Arg219; d, Tyr252-Ala316; e, Ser317-Ala378) in interaction with HPlg are deduced. Region a is important in stabilizing the conformation of the SK molecule, and region b is essential for HPlg activation. Region c is required for induction of the conformational changes of HPlg to virgin enzyme. Regions c and d are required for the conversion of HPlg to HPlm in the HPlg.SK equimolar complex. Coordination of regions c, d, and e of SK is essential for a virgin enzyme formation, and coordination of regions b, c, d and e is required for an effective SK-type HPlg activator.

Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.

PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of adenylate kinase (D. C. Fry, D. M. Byler, H. Susi, E. M. Brown, S. A. Kuby, and A. S. Mildvan, 1988 Biochemistry 27, 3588-3598), beta, gamma-Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety approaching the epsilon-methylene group of K22 in the GX4GKT consensus sequence, in agreement with the X-ray structure of the Mg2+ AMPPNP complex of F1ATPase.

Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease.

By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at subsite 3 [Weber, D.J., Gittis, A.G., Mullen, G.P., Abeygunawardana, C., Lattman, E.E., Mildvan, A.S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5'-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D.J., Gittis, A.G., Mildvan, A.S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5'-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5'-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5'-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those with a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutational tests of the NMR-docked structure of the staphylococcal nuclease-metal-3',5'-pdTp complex.

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.

Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman spectra of horseradish peroxidase and bovine liver catalase compound I species. Evidence for predominant 2A2u pi-cation radical ground state configurations.

The nature of the porphyrin pi-cation radicals in the horseradish peroxidase and bovine liver catalase (BLC) compound I species have been investigated by studying their resonance Raman spectra. A variety of laser excitation and sample interrogation procedures have been employed in order to minimize previously documented problems arising from photoinduced conversions. With Soret band excitation, the spectra obtained for both species resemble that of a compound II-like photoproduct unless the samples are excited with residence times in the microsecond regime with very low (approximately 1 milliwatt) powers. When these precautions are taken, spectra attributable to the compound I species themselves are obtained. The spectrum for horseradish peroxidase compound I is similar to that reported by Paeng and Kincaid (Paeng, K.-J., and Kincaid, J. R. (1988) Am. Chem. Soc. 110, 7913-7915) using a similar approach. Both horseradish peroxidase and BLC compound I exhibit frequency shifts relative to their compound II species that are in the direction observed for model pi-cation radicals with predominant 2A2u character. The magnitudes of these shifts are smaller than those observed for heme models that lack aromatic axial ligands, but agree well with those observed on formation of the compound I analog of N alpha-acetyl microperoxidase-8 that has His as a proximal ligand. This observation is consistent with partial delocalization of the radical density onto the proximal His-170 and Tyr-357 ligands in horseradish peroxidase and BLC, respectively. The strong ligand field provided by these ligands on the proximal side and oxo ligand on the distal side of the heme group is apparently sufficient to reverse the 2A1u radical ground state preference observed for heme-like porphyrin species (e.g. octaethylporphyrins) with weak axial fields. Enhancement of several bands assigned to the Tyr-357 ligand has also been observed for BLC compound I with 406.7-nm excitation. This is attributed either to resonance with a tyrosinate----Fe(IV) charge transfer band or to the coupling provided by radical spin delocalization onto the tyrosinate ligand.

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II.

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.