Alkyl sulfates and alkyl aryl sulfonates as inhibitors of penicillinase]

The kinetics of inhibition of penicillinase of Bac. licheniformis 749/c by various alkyl-sulphates (with a long hydrocarbon chain from C8 to C16), n-toluolsulphoacid and alkylbenzol-sulphonate (R-C12--C16) was studied. The inhibition rate increased with elongation of the alkyl radical and rising of the inhibitor concentraiton. This means that the determining factor in inhibition process was hydrophobic interaction of the alkyl chains and not the electrostatic interaction with the enzyme. In-vitro experiments with penicillinase-producing strains of staphylococcus showed that non-bactericidal concentrations of the alkylsulphates and alkylben zolsulphonates increased the effect of benzylpenicillin, ampicillin and methicillin. The highest increase in the antibiotic activity, as well as in the enzyme inhibition was observed with respect to the compound with the hydrocarbon chain fron C12 to C16. The increase in the activity of methicillin against the staphylococcal strains resistant simultaneously to benzylpenicillin, ampicillin and methicillin was indicative of possible using of the above surface active substances as inhibitors of the realization process of various mechanisms of penicillin resistance.     

Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme-catalysed conversion of D-Tyr-Val-Gly to D-Tyr-Val-NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the alpha-H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non-peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates. The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide-amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co-chromatography in three chromatographic systems. The results demonstrate that the enzyme-catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.     

Effect of surface-active antiseptics on plasmid transduction of drug resistance and the transducing bacteriophages of staphylococci]

Anionic surface active substances (SAS), such as sodium alkylsulfates, iodonate, sulfanol NP-3 used in subbacteriostatic concentrations lowered at least 100 times the intensity of the erythromycin resistance plasmid in vitro on mixed cultivation of the staphylococcal cells of the donor (strain 8325/11 de) and the recipient (strain 825-1). The cationic SAS, i. e. roc cal, chlorhexidine had no such capacity. The above anionic and cationic SAS had an antiphage effect with respect to the transducing staphylococcal bacteriophages of the serological group B (80, 85, 52A, 53). Such an effect (on the example of sodium alkylsulfates) increased with prolongation of the alkyl radical from C8 to C14. A decrease in the transduction intensity of the erythromycin resistant plasmid in staphylococci was observed in the presence of the anionic SAS either possessing (alkylsulfates, iodonate) or not (sulfonol NP-3) the antiphage activity.     

Inhibitory effect of polyelectrolytes on oligomeric enzymes

The effect of polyelectrolytes on the stability and catalytic characteristics of oligomeric enzymes--pig muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH)--was studied by fluorescent spectroscopic and steady state kinetic methods. It was shown that the binding of negatively charged polyelectrolytes--polystyrene sulfonate, polymethacrylate, and polyphosphate--destroys the tertiary and partially the secondary structure of LDH and GDH, resulting in their complete inactivation at pH < 7. The concentrations of polyelectrolytes needed for inhibition of the enzymes were in this case by two or more orders of magnitude lower than the corresponding concentrations for monomers--toluene sulfonate, methacrylate, and phosphate. The affinity of the substrate (pyruvate) for LDH did not vary in the presence of the polyelectrolytes, but the inhibition was removed by excess of substrate. We propose that the oligomeric state of enzymes causes polyelectrolytes to act on them in a special manner, this special effect differing significantly from the effect of polyelectrolytes on monomeric enzymes. The effect consists in that polyelectrolytes cleave the oligomeric structure of the enzymes, this "cleaving" effect being higher the greater the hydrophobicity of the polyelectrolyte chain.     

Prostaglandin H synthase kinetics. The effect of substituted phenols on cyclooxygenase activity and the substituent effect on phenolic peroxidatic activity.

A series of p- and m-substituted phenols were examined for their effect on the cyclooxygenase activity of prostaglandin H synthase in 0.1 M phosphate buffer at pH 8.0 and 25.0 +/- 0.1 degrees C. A biphasic response was observed. At low concentrations phenols stimulate, but at higher concentrations inhibit, cyclooxygenase activity. Both enhancement and inhibition are increased by phenolic substituents which are electron-donating, quantified by Hammett sigma constants, and hydrophobic, quantified by Hantsch tau constants. The same series of substituted phenols was also reacted with compound II of prostaglandin H synthase at 4.0 +/- 0.5 degrees C. The compound II data fit the Hammett rho sigma equation; no hydrophobicity factors are required. Phenols inhibit cyclooxygenase activity by interfering with the binding of arachidonic acid to compound I and by competing directly with arachidonic acid as reducing substrates for compound I. Phenols stimulate cyclooxygenase activity by acting as reducing substrates for compound II, thereby accelerating the peroxidatic cycle. Phenols also protect the enzyme from self-catalyzed inactivation, most likely by removing the free radical of prostaglandin G2 by reducing it to prostaglandin G2. Kinetic parameters Km and kcat for cyclooxygenase activity were determined in the presence of phenols. Identical values of Km (15.3 +/- 0.5 mM) and kcat (89 +/- 2 s-1) were obtained regardless of which phenol was employed. Therefore these represent the true Km and kcat values for cyclooxygenase activity.     

Bacillus licheniformis 749-C plasma membrane penicillinase, a hydrophobic polar protein

Plasma membrane penicillinase from Bacillus licheniformis 749/C is hydrophobic in nature, although it is virtually identical to its riydrophilic exoenzyme counterpart in amino acid composition and sequence. Unlike the exoenzyme, however, the purified membrane enzyme retains [³³P]phosphate and [³H]glycerol. By isoelectricfocusing the membrane enzyme is more acidic than the exoenzyme; it has a lower mobility in SDS gel electrophoresis, consistent with the presence of a very hydrophobic moiety. Unlike the exoenzyme, which binds no taurodeoxycholate, the membrane enzyme binds 10 molecules tightly and approximately 37 molecules in the presence of excess taurodeoxycholate (0.1% solution). The membrane enzyme is identical to the exoenzyme in its reaction with antibodies to exopenicillinase as determined by a radioimmune inhibition assay and immunodiffusion in agar. Heat stability studies indicate a slightly less stable conformation for the membrane enzyme, but this difference largely disappears in the presence of antibody to the exoenzyme. Conversion of membrane enzyme to exoenzyme has been achieved by brief treatment with trypsin, or by incubation of impure preparations at pH 9.0 in 25% potassium phosphate.Since the two forms of penicillinase are very similar in conformation, the hydrophobicity of the membrane form of the enzyme would seem to derive from combination with a hydrophobic moiety, probably phospholipid.     

Scavenging of OH radicals produced in the sonolysis of water

The yield of hydrogen peroxide in the sonication of argon-saturated water was studied in the presence of various solutes. The efficiency of OH radical scavenging is expressed by the reciprocal value of C 1/2, the solute concentration at which the H2O2 yield is decreased by 50 per cent. C 1/2 ranges over several orders of magnitude. It is not related to the specific reactivity towards OH in homogeneous solution. However, it is correlated to the hydrophobicity of the solutes. The competition of I- and a second solute for OH was also studied. The competition between I- and HCO2- follows similar kinetics as in homogeneous solution. However, many other solutes compete in the manner which would be expected if radical scavenging occurred in different phases. The effects are explained in terms of OH radical formation in gaseous argon bubbles, combination of OH radicals to form H2O2 in an interfacial area, and enrichment of hydrophobic solutes in the bubbles.     

The action of acridine derivatives on penicillinase production by staphylococcus aureus]

A study was made of a possibility of inhibition of biosynthesis of penicillinase in Staph. aureus by acridine derivatives. Acetone preparations of penicillinase were obtained from the cultures of staphylococcus strains 16/160 and 8325 (p11(147) pen 1220) grown in the presence of various subbacterial concentrations of acridine derivatives. The activity of the enzyme was studied in experiment and control by the microiodometric method. Acriflavine and proflavine inhibited the penicillinase biosynthesis from the 4th hour of growth, and rivanol, acrichine, acridines No. 27 and 37--from the 12th hour of the culture growth.     

Bacteriophage enzymes for the prevention and treatment of bacterial infections: stability and stabilization of the Streptococcus pyogenes cell lysing enzyme

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the mycelial polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its semiinactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.     

Bacteriophage enzymes for the prevention and treatment of bacterial infections: Stability and stabilization of the enzyme lysing <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> cells

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the micellar polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), ture), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its half-inactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.     

Toward the development of metal-free synthetic nucleases: cleavage of a model substrates by 1,4-diazabicyclo[2.2.2]octane derivatives

Artificial ribonucleases of A(n)BCL series were synthesized by solid-phase method. They consist of a hydrophobic alkyl radical A (n = 3-12 carbon atoms), an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalytic domain" C (histidine residue) and a "linker" L that joins the domains B and C. The effect of the alkyl radical on the catalytic properties of the chemical catalyst was studied using three activated phosphate ester substrates: p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate, and thymidine-3'-p-nitrophenyl phosphate.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Inhibition of the 3 beta-hydroxysteroid oxidoreductase of Pseudomonas testosteroni by steroids.

55 Steroids of the estratriene and androstane type with substituents in pos. 16 alpha, 17 alpha or 17 beta were tested for inhibition of the 3beta-hydroxysteroid oxidoreductase of Pseudomonas testosteroni. Estratrien-3-ols were strong and competitive inhibitors (Ki less than 1 micron). Substituents in pos. 16 alpha of estradiol influenced the inhibitory activity distinctly. Substituents in 17 alpha- or 17 beta-position were of slight influence. 3-Methoxy estratrienes gave no inhibition of the enzymic 3 beta-OH-dehydrogenation. The 4-unsaturated 3-oxo-steroids tested were moderate inhibitors (Ki 2.4-70 micron). The activity was slightly influenced by 17 alpha-substituents. It was increased by 10 beta-substituents in the order H less than CH3 less than N3. The inhibition test can be used to select and eliminate very strong synthetic inhibitors, which are known to disturb the metabolism of steroid hormones.     

In Vitro Stimulation of Protein Kinase C by Melatonin

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca²⁺ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca²⁺ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [³H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [³H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca²⁺. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca²⁺ dependent proteins.     

Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A

The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH2CO2CH(CH2CH3)CO2H (2)) by bovine pancreatic carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) have been studied over the substrate concentration range 10(-4)-10(-1) M at pH 7.5, 25 degrees C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH3, CH3CH2 and (CH3)3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES2 complex and parameters for the second-degree rate equation v/E = (kapp2 S + kapp3 S2/KappSS)/(KappS + S + S2/KappSS) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH3 and Y = cyclo-C6H11 the following linear correlations were observed: -log KappS = 0.82 pi + 1.32 and log kapp2/KappS = 0.71 pi + 5.81 (pi is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. KappSS also depends on the hydrophobicity of Y but no quantitative correlation is obvious. Thus the Y unit of 2 is involved in a hydrophobic interaction with the enzyme when 2 binds at both the catalytically productive and inhibitor sites. Parameters for the enzymic hydrolysis of the esters YCONHCH2CO2CH(CH2CH(CH3)2)CO2H (3) (Y = C6H5(CH2)n (n = 0, 1, 2)) are also presented. Pronounced nonproductive 1: 1 enzyme.substrate complex formation is observed for each of 2: Y = C6H5(CH2)n (n = 2, 3) and 3: Y = C6H5(CH2)2. Hippurate anion is shown to be an uncompetitive inhibitor (Ki = 12 mM) for the hydrolysis of 2: Y = (CH3)3C. Data are now available which can only be interpreted in terms of at least three enzymic sites being available for hydrophobic interactions with ester substrate molecules.     

Membrane fusogenic lysine type lipid assemblies possess enhanced NLRP3 inflammasome activation potency

Lysine (K) type cationic lipid with a propyl spacer and ditetradecyl hydrophobic moieties composing liposomes, K3C14, previously studied for gene delivery, were reported to activate the NLRP3 inflammasomes in human macrophages via the conventional phagolysosomal pathway. In this study, K3C16, a propyl spacer bearing lysine type lipids with dihexadecyl moieties (an extension of two hydrocarbon tail length) were compared with K3C14 as liposomes. Such a small change in tail length did not alter the physical properties such as size distribution, zeta potential and polydispersity index (PDI). The NLRP3 activation potency of K3C16 was shown to be 1.5-fold higher. Yet, the toxicity was minimal, whereas K3C14 has shown to cause significant cell death after 24 h incubation. Even in the presence of endocytosis inhibitors, cytochalasin D or dynasore, K3C16 continued to activate the NLRP3 inflammasomes and to induce IL-1β release. To our surprise, K3C16 liposomes were confirmed to fuse with the plasma membrane of human macrophages and CHO-K1 cells. It is demonstrated that the change in hydrophobic tail length by two hydrocarbons drastically changed a cellular entry route and potency in activating the NLRP3 inflammasomes.     

Synthesis and biological evaluation of sialic acid derivatives containing a long hydrophobic chain at the anomeric position and their C-5 linked polymers as potent influenza virus inhibitors

Conversions of the C-5 acetamide group in sialic acid into two kinds of C=C double bond substituents were accomplished under Shotten-Baumann conditions. The polymerizable glycomonomers also contain a hydrophobic chain or hydroxyl group at the anomeric position. Radical polymerizations of the fully protected glycomonomers were carried out with acryl amide in the presence of ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), followed by deprotection to furnish water-soluble glycopolymers. The activities of the deprotected glycopolymers and glycomonomers against human influenza viruses (H1N1 and H3N2) and avian influenza virus (H5N3) were evaluated. Biological evaluations showed that the glycomonomers containing a long hydrophobic chain at the anomeric position had both hemagglutination and neuraminidase inhibitory activities.     

Self-assembled nanoparticles of dextrin substituted with hexadecanethiol

The amphiphilic molecule dextrin-VA-SC16 (dexC16) was synthesized and studied in this work. DexC16 has a hydrophilic dextrin backbone with grafted acrylate groups (VA) substituted with hydrophobic 1-hexadecanethiol (C16). A versatile synthetic method was developed allowing control of the dextrin degree of substitution with the hydrophobic chains (DSC16, number of alkyl chains per 100 dextrin glucopyranoside residues). Materials with different DSC16 were prepared and characterized using 1H NMR. DexC16 self-assembles in water through association of the hydrophobic alkyl chains, originating nanoparticles. The nanoparticles properties were studied by dynamic light scattering (DLS), fluorescence spectroscopy, and atomic force microscopy (AFM).     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.