Accelerated Adsorption of Bacteriophage T5 to Escherichia coli F, Resulting from Reversible Tail Fiber-Lipopolysaccharide Binding

A dual specificity for phage T5 adsorption to Escherichia coli cells is shown. The tail fiber-containing phages T5(+) and mutant hd-3 adsorbed rapidly to E. coli F (1.2 x 10(-9) ml min(-1)), whereas the adsorption rate of the tail fiber-less mutants hd-1, hd-2, and hd-4 was low (7 x 10(-11) ml min(-1)). The differences in adsorption rates were due to the particular lipopolysaccharide structure of E. coli F. Phage T4-resistant mutants of E. coli F with an altered lipopolysaccharide structure exhibited similar low adsorption for all phage strains with and without tail fibers. The same held true for E. coli K-12 and B which also differ from E. coli F in their lipopolysaccharide structures. Only the tail fiber-containing phages reversibly bound to isolated lipopolysaccharides of E. coli F. Infection by all phage strains strictly depended on the tonA-coded protein in the outer membrane of E. coli. We assume that the reversible preadsorption by the tail fibers to lipopolysaccharide accelerates infection which occurs via the highly specific irreversible binding of the phage tail to the tonA-coded protein receptor. The difference between rapid and slow adsorption was also revealed by the competition between ferrichrome and T5 for binding to their common tonA-coded receptor in tonB strains of E. coli. Whereas binding of T5(+) to E. coli K-12 and of the tail-fiber-less mutant hd-2 to E. coli F and K-12 was inhibited 50% by about 0.01 muM ferrichrome, adsorption of T5 to E. coli F was inhibited only 40% by even 1,000-fold higher ferrichrome concentrations.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.     

A synthetic heptasaccharide reveals the capability of a monoclonal antibody to read internal epitopes of a polysaccharide antigen.

The binding of the synthetic heptasaccharide,beta-D-Galp-(1----3)-beta-D-Galp-(1----6)-beta-D-Galp-(1 ----6)-beta-D-Galp-(1----6)-beta-D-Galp-1-OCH3 (10) with two monoclonal IgAs of the X24 gene-family has been investigated. The ligand 10 was synthesized by silver triflate mediated coupling of O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----3)-O-(2,4,6,-t ri-O-benzoyl-beta-D-galactopyranosyl)-(1----3)-2,4,6-tri-O-benzoyl-alpha -D-galactopyranosyl chloride (5) to the benzoylated, all-beta-(1----6)-linked methyl galactotetraoside 13, having O-6(4) free, followed by debenzoylation of the formed, fully protected methyl galactoheptaoside. The blockwise synthesis of the nucleophile 13 from readily available monosaccharides, and the synthesis of 5 from the corresponding beta-1-O-benzoylated trisaccharide, is also described. Heptasaccharide 10 binds with the (1----6)-beta-D-galactan-specific monoclonal antibodies X-24 and J539 with essentially the same Ka-values (5.4 x 10(5) M-1 and 6.4 x 10(5) M-1, respectively) as does the methyl beta-glycoside of all-beta-(1----6)-linked galactotetraose 14 (5.7 x 10(5) M-1 and 5.9 x 10(5) M-1, respectively). Of the series of homologous oligosaccharides studied previously (di- through a hexa-saccharide), 14 was found to show the highest affinity of interaction with both these immunoglobulins. The beta-(1----3)-linked galactotriose, which forms the bulky terminus of 10, does not appear to bind to these IgA. Thus, the observation that the affinity of 10 is the same as that of 14 confirms that these immunoglobulins bind internal tetrasaccharide sequences of the antigenic (1----6)-beta-D-galactopyranan.     

'Receptor-independent' infection of Mu G(−) phage in Escherichia coli K-12

Abstract Bacteriophage Mu with its invertible G segment in G(−) orientation does not make plaques on Escherichia coli K-12, due to the absence of a suitable lipopolysaccharide receptor. Plaques formed by Mu G(−) were found, however, when the infected E. coli K-12 strain harbours a plasmid with the cloned DNA inversion function Gin which converts the infecting G(−) phage to G(+). Under overproducing conditions, where Gin expression is placed under the control of the tac promoter, the infectivity of Mu G(−) can be estimated as approximately 1% of that in the presence of the receptor. Furthermore, interaction of Mu G(−) with the E. coli K-12 cell wall leads to interference with the plating of a Mu G(+) variant which has the new phenotype Pen⁻ (penetration-negative).     

Blocking of human immunodeficiency virus infection depends on cell density and viral stock age.

Quantitative infectivity assays were used to study how the blocking activity of soluble CD4 (sCD4) is affected by sCD4 concentration, target cell density, and viral stock age. During incubation with 20 nM sCD4, human immunodeficiency virus type 1 (HIV-1) stocks underwent irreversible inactivation. In contrast, inactivation with 2 nM sCD4 was almost entirely reversible. At lower sCD4 concentrations (less than or equal to 2 nM) and target cell densities of 6.25 x 10(4) ml-1, sCD4 blocking activity for HIV-1 gave a gp120-sCD4 association constant (Kassoc) of 1.7 x 10(9) M-1, which agrees with chemical measurements. At the higher density of 1.6 x 10(7) cells ml-1, however, the blocking activity was 20-fold less. During incubation of HIV-1 stock optimized for infectivity by rapid harvest, sCD4 blocking activity increased 20-fold during a 3-h window. These results show that competitive blocking activity depends strongly on target cell density and virion age. Thus, unappreciated variations in HIV stocks and assay conditions may hinder comparisons of blockers from laboratory to laboratory, and the age of HIV challenge stocks may influence studies of drug and vaccine efficacy. The results also suggest that blocking of viral particles in lymphoid compartments will require very high competitive blocker concentrations, which may explain the refractory outcomes from sCD4-based drug trials in humans.     

A new electro-optical approach to rapid assay of cell viability

A new electro-optical (EO) approach was developed and applied to rapidly assay cell viability by using phage M13K07. Since phage M13K07 can replicate only in living bacteria and cannot replicate in the presence of inhibitors, the difference between the EO signals obtained in the presence and absence of the phage can be used as an important factor for evaluating cell viability. Variation in the electrophysical parameters of Escherichia coli XL-1 during its interaction with phage M13K07 was studied under exposure of the cells to various inhibitors of cellular metabolism. Significant changes in the EO signal were found during incubation of living E. coli cells with phage M13K07. At the same time, no changes were recorded during cell incubation with the phage after pretreatment of E. coli XL-1 cells with sodium azide, carbonyl cyanide 3-chlorophenyl hydrazone, chloramphenicol, and kanamycin. This finding can be explained by the decrease in the number of living cells in the culture after preliminary incubation with the chemical agents, and it was confirmed by colony counts by conventional plating onto solid LB medium before and after treatment of the cells with the inhibitors. The EO approach can be used as a rapid method for evaluation of the inhibitory effects of various chemical agents and drugs, and it has the potential for the study of the molecular mechanisms underlying cell death.     

Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.     

Heparin modulation of human plasma kallikrein on different substrates and inhibitors

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.     

Transducing phages of Pseudomonas aeruginosa related to phage F116L

New phages K104 and B26, which are relative to F116L by a number of biological characters, appeared to show general transducing activity. Phage K104 transduces all tested markers with higher frequency than the phage B26. Linkage of the bacterial markers pair ilv202--met28 durspectively. When recipient bacteria lysogenic for phages K104 and B26 are used, frequencies of transduction by phage F116L are decreased. In the presence of F116L prophage the frequency of transduction by phage B26 is 10-fold increased. Phages B26 and F116L do not grow on bacteria lysogenic for these phages. Phage F116L does not grow on the lawn of bacteria, lysogenic for phage K104, while phage B26 grows on the same lawn with the efficiency of plating about 10(-2).     

A phasmid shuttle vector for the cloning of complex operons in Salmonella

Phasmid (phage plasmid hybrid) P4 vir1 can be propagated in Escherichia coli as a helper-dependent lytic phage, as a plasmid, or as a prophage. On the basis of an understanding of these modes of propagation, derivatives of P4 have been constructed for use as cloning vectors. In this report we demonstrate that phasmid P4 (i) will propagate as a helper-dependent lytic phage and as a plasmid in Salmonella spp. and (ii) can be used as a high efficiency phage shuttle vector for the reversible transfer of cloned genes between Salmonella spp. and E. coli. For both E. coli and Salmonella spp., P4 phage-mediated gene transfer proved to be only 10-fold lower than plaquing efficiency. For the case of Salmonella spp., this frequency is ca. 10(4)-fold more efficient than is typically found for the transformation of DNA molecules. The usefulness of this cloning vector system for analyses of pathogenic virulence factors is demonstrated by the cloning and expression of both the P pilus adhesin operon and the hemolysin operon of uropathogenic E. coli.     

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.     

Isolation and partial characterization of a bacteriophage T5 mutant unable to induce thymidylate synthetase and its use in studying the effect of uracil incorporation into DNA on early gene expression.

A mutant of phage T5 which is unable to induce thymidylate synthetase was isolated. T5 thy mutants synthesized less DNA than did wild-type T5, and the burst size of progeny phage was correspondingly reduced two- to threefold in thy+ Escherichia coli. No DNA or progeny phage were made in E. coli thy hosts grown in the absence of exogenous thymine. When the T5 thy mutation was recombined with a T5 dut mutation (unable to induce dUTPase), replication resulted in progeny which contained significant amounts of uracil in their DNA, and these phage failed to produce plaques unless the plating host was deficient in uracil-DNA glycosylase. T5 phage containing various amounts of uracil in their DNA were prepared and used to determine the effect of uracil on the induction of the early enzyme dTMP kinase. The presence of uracil in the parental DNA increased the rate of induction of this enzyme by about 2.5-fold. The T5 thy gene was mapped and is located near the T5 frd gene on the B region of the T5 genome.     

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.     

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

The selective inhibition of thrombin by peptides of boroarginine

Peptides containing alpha-aminoboronic acids with neutral side chains are highly effective reaction intermediate analog inhibitors of the serine proteases leukocyte elastase, pancreatic elastase, and chymotrypsin. A protocol has been developed for the synthesis of peptides containing alpha-aminoboronic acids with a basic, 3-guanidinopropyl side chain (boroArg) to extend the range of these compounds to trypsin-like proteases. Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-OH were prepared as inhibitors of thrombin based on earlier observations that it has a high affinity for this sequence. All three boronic acids are highly effective, slow-binding inhibitors of thrombin, inhibiting it with final inhibition constants and association rates of: 41 pM, 5.5 x 10(6) M-1 s-1; 3.6 pM, 9.3 x 10(6) M-1 s-1; less than 1 pM, 8.0 x 10(6) M-1 s-1, respectively. Comparison of their binding at equilibrium to thrombin, plasma kallikrein, factor Xa, plasmin, and two-chain tissue plasminogen activator has shown that all three inhibitors have at least 2 orders of magnitude greater affinity for thrombin, with the exception of the acetyl derivative which has a 40-fold greater affinity for thrombin than kallikrein. The boroarginine peptides are effective in inhibiting the action of thrombin in rabbit plasma against its physiological substrates. Activated partial thromboplastin time was significantly prolonged in vitro by all of the inhibitors at concentrations of 50-200 nM. Prolongations of activated partial thromboplastin time were also observed in rabbits after intravenous (40-80 micrograms/kg or subcutaneous (0.20-2 mg/kg) injections of Ac-(D)Phe-Pro-boroArg-OH. Results indicate that this new class of synthetic thrombin inhibitors may be clinically useful as antithrombotic agents.     

Mg2+ dependence of guanine nucleotide binding to tubulin

The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1.     

Uptake and destruction of 125I-CSF-1 by peritoneal exudate macrophages

The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.