Sporadic Distribution of tRNACCUArg Introns among α-Purple Bacteria: Evidence for Horizontal Transmission and Transposition of a Group I Intron

A group I intron interrupts the tRNA_CCU^Arg gene of the α-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173–176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of α-purple bacteria. Of 10 newly identified tRNA_CCU^Arg genes, we found only two that contained an intron homologous to that of the Agrobacterium tRNA_CCU^Arg intron. This restricted and scattered distribution of the tRNA_CCU^Arg intron among α-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA_UAA^Leu introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293–298, 1997) and α-purple tRNA_CCU^Arg introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA_UAA^Leu introns.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 μM concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-α-trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 μM concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase α₂β₂ multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium α-glycerophosphate (Na₂GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of βE109D and βD305A mutations, on K_eq for the conformational equilibria were determined. The l-Ser external aldimine-aminoacrylate equilibrium (K_eq = [external aldimine]/[aminoacrylate]) has the largest value with Na⁺ (0.12), followed by K⁺ (0.04), Li⁺ (7.6 × 10⁻⁴), Rb⁺ (4.3 × 10⁻⁴), NH₄⁺ (2.3 × 10⁻⁴), no cation (2.0 × 10⁻⁴) and Cs⁺ (1.6 × 10⁻⁵). α-Site ligands, Na₂GP and IAG, have modest 3- to 40-fold effects on K_eq in the direction of aminoacrylate, but BZI in the presence of Na⁺ gives a low value of K_eq comparable to that obtained with Cs⁺. There is no additivity of free energy for Na₂GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of ΔV_o range from −126 mL/mol for the Na⁺ complex to −204 mL/mol for the Na⁺ complex with BZI. The βD305A mutation changes the K_eq by a factor of at least 10⁵ (26.7 kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both ΔV_o and ΔS for the l-Ser external aldimine-aminoacrylate equilibrium for βD305A Trp synthase, consistent with a large decrease in solvation accompanying the conformational change in βD305A Trp synthase relative to wild-type Trp synthase. The βE109D mutation has more modest but significant effects on K_eq, which differ with the ligand, ranging from 40-fold for GP to 2200-fold for BZI, even though βGlu-109 is not directly involved in allosteric communications. The effect of GP on the external aldimine-quinonoid intermediate equilibrium of the Trp synthase-l-Trp complex is similar to that of GP on the Trp synthase-l-Ser external aldimine-aminoacrylate equilibrium. These results have allowed a quantitative comparison of the allosteric effects of ligand and mutations in Trp synthase. These allosteric effects are finely tuned to control the synthesis of l-Trp without resulting in substrate or product inhibition.     

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

The production and characterization of monoclonal antibodies against enkephalins

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu⁵-enkephalin that were highly specific for Leu⁵- and Met⁵-enkephalin. AD4 exhibited almost equal reactivity with either Leu⁵- or Met⁵-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met⁵-enkephalin. The IC₅₀ of these monoclonal antibodies were approximately two orders of magnitude greater than the IC₅₀ a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies.The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1–6 or 1–13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.     

New advances in the study on the interaction of [Cr(phen)2(dppz)]3+ complex with biological models; association to transporting proteins

The present study reports a detailed investigation with the interaction of [Cr(phen)₂(dppz)]³⁺ with serum albumins, the key protein for the transport of drugs in the blood plasma, which allows us to understand further the role of [Cr(phen)₂(dppz)]³⁺ as sensitizer in Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)₂(dppz)]³⁺, (dppz = dipyridophenazine and phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly with bovine and human serum albumins (BSA and HSA) with an intrinsic binding constants, K_b, of (1.7 ± 0.3) × 10⁵ M^− 1 and (2.2 ± 0.3) × 10⁵ M^− 1 at 295 K, respectively. The interactions of serum albumins with [Cr(phen)₂(dppz)]³⁺ were assessed employing fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy. The serum albumins-[Cr(phen)₂(dppz)]³⁺ interactions caused conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the albumin (BSA or HSA) bound to the Cr(III) complex decreased, suggesting that perturbation around the Trp 214 residue took place. The analysis of the thermodynamic parameters ΔG, ΔH, ΔS indicated that the hydrophobic interactions played a major role in both BSA-Cr(III) and HSA-Cr(III) association processes. The binding distances and transfer efficiencies for BSA-Cr(III) and HSA-Cr(III) binding reactions were calculated according to the Föster theory of non-radiation energy transfer. All these experimental results suggests that [Cr(phen)₂(dppz)]³⁺ binds to serum albumins, by which these proteins could act as carriers of this complex for further applications in PDT.     

Synthesis, characterization, X-ray crystallography, and cytotoxicity of a cymantrene keto carboxylic acid for IR labelling of bioactive peptides on a solid support

Cym-CO-CH₂-CH₂-COOH was prepared in good yield by Friedel-Crafts reaction of cymantrene (Cym, CpMn(CO)₃) with succinic anhydride for the IR labelling of peptides and fully characterized, including an X-ray structure analysis (monoclinic space group P2(1)/n, a = 5.727(3) Å, b = 19.865(9) Å, c = 10.518(5) Å, β = 91.211(9)°). The compound was isolated in pure form without the need for chromatographic work-up and subsequently used for solution-phase synthesis of a bioconjugate with phenylalanine methyl ester to allow a complete spectroscopic characterization of this model system. The cymantrene keto carboxylic acid also turned out to be a very robust marker in automated microwave-assisted solid phase peptide synthesis (SPPS). [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was prepared on a Wang resin and labelled with the cymantrene derivative on the solid support under microwave irradiation in all steps. The metal-carbonyl marker stayed intact during cleavage from the resin with concentrated trifluoroacetic acid. After simple precipitation and lyophilization, the cymantrene-enkephalin bioconjugate could be obtained in analytically pure form without the need of HPLC purification. As required, the compound is non-cytotoxic against MCF-7 cells at up to 100 μM. This protocol thus allows one to introduce organometallic IR spectroscopic labels to peptides in a very straightforward way.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Effect of dioxygen on copper(II) binding to α-synuclein

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (K_d(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M^∗DV(F/W), where M^∗ = methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a Cu^II-Trp/Phe (cation-π) interaction.     

Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

The β₁-adrenergic receptor (β₁AR) is the predominant βAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human β₁AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293_i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn¹⁵. Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg³¹ and Leu³² and Pro⁵² and Leu⁵³, which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the βAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg³¹–Leu³² cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface β₁ARs.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter--trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.     

Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells

Neurotensin (NT) analogs, modified at Arg⁸ and Arg⁹, were used to assess the role of Arg in NT binding to mast cells. [D-Arg⁸]- and [D-Arg⁹]-NT bound 4–5 times better than NT, whereas [D-Arg^8,9]-NT had the same binding affinity as NT. Binding of [Ala⁸]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala⁹]-NT had 32% binding. C-terminal peptides, NT_8–13 and NT_9–13, had about 65% binding. These data suggest that Arg⁸ plays a greater role than Arg⁹ in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys^2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.     

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB₁) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB₁ receptor agonist [³H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC₅₀s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [³H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC₅₀ at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [³H]CP-55940 binding assay were positively correlated with inhibition of CB₁ receptor agonist-stimulated binding of [³⁵S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [³H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [³H]SR141716A showed that phthalates reduce the B_max of radioligand without changing its K_d. DnBP and nBBP also rapidly enhanced the dissociation of [³H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB₁ receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB₁ receptor-dependent behavioral and physiological outcomes in the whole animal.     

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an M_r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000–1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg⁷⁰⁹/Arg¹⁰¹⁸/Arg¹⁵⁴⁵) mutated to glutamine (fV^Q3), a mutant fV molecule with region 1000–1008 deleted (fV^ΔB9), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV^ΔB9/Q3). The recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV^Q3 was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K_D(app) values for fV^ΔB9 (0.9 nm), fVa^ΔB9 (0.4 nm), and fV^ΔB9/Q3 (0.7 nm) were similar to the affinity of fVa^WT for fXa (0.3 nm). Two-stage clotting assays revealed that although fV^Q3 was deficient in its clotting activity, fV^ΔB9/Q3 had clotting activity comparable with fVa^WT. The k_cat value of prothrombinase assembled with fV^ΔB9/Q3 was minimally affected, whereas the K_m value of the reaction was increased 57-fold compared with the K_m value obtained with prothrombinase assembled with fVa^WT. These findings strongly suggest that amino acid region 1000–1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.