The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.     

Adsorption and infectivity of human immunodeficiency virus type 1 are modified by the fluidity of the plasma membrane for multiple-site binding

Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for "multiple-site binding," we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV-1) and a pseudotyped luciferase-expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post-attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 micro M of anti-CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple-site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple-site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 micro g/ml of anti-HLA-II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti-HLAII antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple-site binding.     

Real-time observations of individual macaque sperm undergoing tight binding and the acrosome reaction on the zona pellucida

Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was added. Capacitated sperm were added together with Alexa-SBTI, a probe for acrosin that can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. Tight binding was observed after 16 sec as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, C-shaped waveform. The first signs of the acrosome reaction were detected within 11 sec of tight binding. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of those that were tightly bound. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects the initial attachment or docking of sperm to the zona, a step that may align or recruit one or more specific zona receptors to be responsible for mediating the acrosome reaction.     

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.     

Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F-2044.1

Hydroxides of magnesium and zinc, aluminum oxide, zinc phosphate, and co-precipitated Ca3(PO4)2 and Mg(OH)2 were efficient in binding extracellular glucose oxidase (GO) of P. adametzii LF F-2044.1 in a culture liquid filtrate (CLF). Basic Al2O3 was the most appropriate adsorbent for GO isolation from the CLF of the fungus. A GO isolation method was developed, which allowed for obtaining an enzyme with a high degree of purification. Spectral properties of the enzyme, its catalytic activity, and stability were characterized. The GO of P. adametzii LF F-2044.1 exhibited high pH stability, retaining activity within the range 4.5-9.0. The rate that GO-catalyzed D-glucose oxidation increased as the temperature increased (up to approximately 60 degrees C). The catalytic activity and thermal stability of GO depended on its concentration in the medium. Under optimum conditions, the fractions GO-1 and GO-2 were characterized by KM values of 1.56 x 10(-2) and 2.19 x 10(-2) M, respectively; the corresponding values of kcat equaled 235.1 and 318.2 s(-1).     

A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.     

Distinct cellular receptor interactions in poliovirus and rhinoviruses

Receptor binding to human poliovirus type 1 (PV1/M) and the major group of human rhinoviruses (HRV) was studied comparatively to uncover the evolution of receptor recognition in picornaviruses. Surface plas- mon resonance showed receptor binding to PV1/M with faster association and dissociation rates than to HRV3 and HRV16, two serotypes that have similar binding kinetics. The faster rate for receptor association to PV1/M suggested a relatively more accessible binding site. Thermodynamics for receptor binding to the viruses and assays for receptor-mediated virus uncoating showed a more disruptive receptor interaction with PV1/M than with HRV3 or HRV16. Cryo-electron microscopy and image reconstruction of receptor-PV1/M complexes revealed receptor binding to the 'wall' of surface protrusions surrounding the 'canyon', a depressive surface in the capsid where the rhinovirus receptor binds. These data reveal more exposed receptor-binding sites in poliovirus than rhinoviruses, which are less protected from immune surveillance but more suited for receptor-mediated virus uncoating and entry at the cell surface.     

Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

The kinetics of the increase in protein fluorescence following the addition of ATP to subfragment-1 (SF-1) and acto-SF-1 have been reinvestigated. The concentration dependence of the rate obtained with SF-1 did not fit a hyperbola and at high ATP concentration, approximately 40% of the signal amplitude was lost due to a fast phase at the beginning of the transient (20 degrees C). At lower temperature (less than or equal to 10 degrees C) the fluorescence transient was biphasic, with a fast phase observed at high ATP concentration. These results indicate that there are two steps in the SF-1 pathway in which there is a change in protein fluorescence. Measurements of ATP binding and hydrolysis by chemical quench-flow methods indicate that the rate of ATP binding is correlated with the fast fluorescence step and hydrolysis is correlated with the slow fluorescence change. The SF-1 mechanism can thus be described as: (formula: see text) where M represents SF-1 and states of enhanced fluorescence are given by M (16%) and M (36% enhancement, relative to SF-1). Step 1 is a rapid equilibrium with K1 approximately 10(3) M-1. Tight binding of ATP occurs in step 2 and the loss of signal amplitude requires k2 greater than or approximately 1500--2000 s-1. The maximum observed fluorescence rate defines the rate of hydrolysis, k3 + k-3 = 125 s-1 (20 degrees C, 0.1 M KCl, pH 7.0). The steps in the mechanism correspond to the Bagshaw--Trentham scheme, with the important difference that the assignment of rate constant is altered. Formation of the acto-SF-1 complex gave a fluorescence enhancement of approximately 14% relative to SF-1. Dissociation of acto-SF-1 by ATP produced a 20--22% enhancement in fluorescence. There was no detectable fluorescence change during dissociation as evidenced by a lag in the fluorescence transient which corresponded to the kinetics of dissociation. The fluorescence change occurred at the same maximum rate as for SF-1 but there was no loss in signal amplitude at high ATP concentration. The kinetics of the fluorescence change corresponded to the rate of ATP hydrolysis, whereas tight ATP binding occurred at a much faster rate in approximate agreement with the rate of dissociation. Thus the fluorescence change in the acto-SF-1 pathway corresponds to step 3 in the SF-1 mechanism. The complete scheme can be described as follows: (formula: see text) where AM represents acto-SF-1. The tight binding step in the SF-1 pathway (k2) is sufficiently fast so that a similar step (k2') in the acto-SF-1 pathway could precede dissociation but the AM-ATP intermediate has not been detected. Following hydrolysis on the free SF-1, actin recombines with M.ADP.Pi or possibly with a second SF-1 product intermediate as proposed by Chock et al. (1976) and the fluorescence returns to the original AM level with product release.     

Leishmania tropica: Biochemical aspects of promastigotes' attachment to macrophages in vitro

The in vitro attachment of promastigotes of Leishmania tropica to mouse peritoneal macrophages was studied under experimental conditions. Parasite binding by macrophages required an intact intracellular physiology as suggested by the action of several metabolic inhibitors. Parasite attachment was inhibited in the presence of azide, fluoride, iodoacetamide, and 2-deoxyglucose, but was affected very little by inhibitors of oxidative phosphorylation, by 2,4,-dinitrophenol and by cyanide. Parasite attachment was not prevented by cycloheximide, an inhibitor of protein synthesis. Successful parasite attachment required the simultaneous presence of magnesium, glucose, and a macromolecular component of fetal calf serum in the extracellular medium. Furthermore, glucose and serum supported parasite binding synergistically. The requirement for extracellular glucose could be replaced by mannose, suggesting that such a requirement is structural rather than metabolic. The active fraction of serum was nondialyzable, heat labile, and precipitable by ammonium sulfate. These various chemical ingredients of the culture medium were required mainly during the interaction of parasites with macrophages. The extracellular and metabolic requirements of successful parasite binding suggest that attachment of parasites to macrophages possess the characteristics of a biochemical reaction which is probably mediated by one or more enzymatic reactions.     

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.     

Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds

Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.     

Rabbit M type phosphoglyceromutase: comparative effects of two thiol reagents antibody reaction and hybridization studies

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.     

Distribution of negatively charged liposomes on rat liver hepatocytes and non-hepatocytes in cell suspension

The in vitro interactions between negatively charged multilamellar liposomes and purified rat liver parenchymal and non-parenchymal cells were studied. The liposomes were labelled with [¹⁴C]cholesterol and contained [³H]methotrexate. For both cell types the time course of liposomal attachment to the cells slowed down gradually after a rapid initial phase lasting ca 90 min. The rate of attachment at 4 °C was 3–7 times lower than that at 37 °C, and the metabolic inhibitors dinitrophenol and iodoacetic acid caused reduction of 20–30%. Up to 45% of the cell-associated liposomal radioactivity could be detached within 1 h incubation with unlabelled liposomes. Whereas liver parenchymal cell suspension seemed to exhibit similar characteristics in vitro as in vivo, the non-parenchymal cells in vitro showed a 20–50-fold reduction in the rate of liposomal attachment compared to in vivo.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca(+2)-independent and distinct from a Ca(+2)-dependent hyaluronan binding activity.

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I-hyaluronan (HA) and internalize it using a coated pit pathway [Biochem J, 257:875-884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I-HA binding to intact LECs at 4 degrees C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I-HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca(+2)-independent HA binding activity increased approximately 743%, while the Ca(+2)-dependent binding activity was enhanced only approximately 46%. Therefore, the Ca(+2)-dependent HA binding activity appears not to be intracellular, whereas the Ca(+2)-independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I-HA at 37 degrees C in 10 mM EGTA or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I-HA in the presence of 10 mM Ca+2, the amount of cell-associated radioactivity increased approximately 20-50-fold. However, this additional cell-associated 125I-HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10 mM EGTA at 4 degrees C. Therefore, the Ca(+2)-dependent cell-associated 125I-HA had accumulated on the cell surface and had not been internalized. From these studies we conclude that LECs have at least two types of specific HA binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.     

Discrimination among rhinovirus serotypes for a variant ICAM-1 receptor molecule

Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinovirus serotypes, including human rhinovirus 14 (HRV14) and HRV16. A naturally occurring variant of ICAM-1, ICAM-1Kilifi, has altered binding characteristics with respect to different HRV serotypes. HRV14 binds to ICAM-1 only transiently at physiological temperatures but forms a stable complex with ICAM-1Kilifi. Conversely, HRV16 forms a stable complex with ICAM-1 but does not bind to ICAM-1Kilifi. The three-dimensional structures of HRV14 and HRV16, complexed with ICAM-1, and the structure of HRV14, complexed with ICAM-1Kilifi, have been determined by cryoelectron microscopy (cryoEM) image reconstruction to a resolution of approximately 10 angstroms. Structures determined by X-ray crystallography of both viruses and of ICAM-1 were fitted into the cryoEM density maps. The interfaces between the viruses and receptors contain extensive ionic networks. However, the interactions between the viruses and ICAM-1Kilifi contain one less salt bridge than between the viruses and ICAM-1. As HRV16 has fewer overall interactions with ICAM-1 than HRV14, the absence of this charge interaction has a greater impact on the binding of ICAM-1Kilifi to HRV16 than to HRV14.     

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.