The Effect of Thermal Treatment on the Catalytic Activity and Biological Properties of Bacillus intermedius Ribonuclease

Bacillus intermedius ribonuclease (binase), which is known to exert a growth-stimulating effect at low concentrations and a genotoxic effect at high concentrations, loses these abilities completely after exposure to 100°C for 10 min, but retains approximately 95% of its catalytic activity and structural integrity. Other types of modification, such as photoinactivation and site-specific mutagenesis, gave rise to enzyme forms with unaltered structure but reduced (sometimes to trace amounts) catalytic activity. Genotoxicity was always proportional to the catalytic activity of the native enzyme, while a notable growth-stimulating effect could be exerted by enzymes with low activity. The loss of biological activity of thermoinactivated binase was related to the increase in the number of negatively charged groups on the enzyme surface, which led to a substantial decline in the adhesive properties of the enzyme.     

Induction of apoptosis of tumor cells by binase

An induction of apoptosis by RNase from Bacillus intermedius (binase) and its mutants characterized with low catalytic activity (Lys26Ala and His101Glu) in human myelogenic erythroleukemia K562 cells, human lung carcinoma A549 cells and human peripheral blood mononuclear cells was studied. For the first time selective apoptogenic effects of binase toward leukemic blood cells was determined. Neither antiproliferative nor apoptotic effects of binase were detected in normal human peripheral blood mononuclear cells. Formation of low molecular weight oligonucleosomal DNA fragments (less than 50 Kb) which are an early marks of apoptosis was registered in solid tumor cells treated by binase. Using mutant RNases it was shown that decrease of catalytic activity to 2.5% of wild type enzyme activity leads to the loss of apoptogenic properties of enzyme. Selective apoptogenicity of binase found towards malignant cells confirmed that antitumor agents based on bacterial RNases could be considered as an alternative to standard chemotherapeutic drugs.     

Induction of Apoptosis in Tumor Cells by Binase

The possibility of inducing apoptosis in K562 myelogenic erythroleukemia cells, A549 lung carcinoma cells, and normal human lymphocytes was studied for Bacillus intermedius RNase (binase) and its mutants Lys²⁶ Ala and His¹⁰¹ Glu with impaired catalytic activity. Selective induction of apoptosis in leukemic blood cells by binase was demonstrated for the first time. Binase did not exert an antiproliferative or proapoptotic effect on peripheral blood lymphocytes of healthy donors. Low-molecular-weight (less than 50 kb in size) oligonucleosomal DNA fragments, which are early markers of apoptosis, were observed in human solid-tumor cells treated with binase. Studies with the binase mutants showed that a decrease in catalytic activity to 2.5% of the level characteristic of the wild-type enzyme deprives binase of its proapoptotic effect. The selective proapoptotic effect of binase on malignant cells provides evidence that bacterial RNases are promising for designing alternative antitumor drugs.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 457–463.Original Russian Text Copyright © 2005 by Zelenikhin, Kolpakov, Cherepnev, Ilinskaya.     

Binase penetration into alveolar epithelial cells does not induce cell death

Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.     

X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 Å resolution. The kinetic parameters of the hydrolysis of substrates of different length (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease of the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.     

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.     

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.     

Exogenous <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RNase (binase) suppresses the reproduction of reovirus serotype 1

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.     

The mechanisms of the manifestation of the genotoxic effects of binase]

The high concentrations of commercial sample of Bacillus intermedius 7P ribonuclease (binase) showed a weak mutagenic effect in Ames test and Ara-test. It has been established that binase induces the SOS-functions of microbial cell and prophage induction. The way of exogenic enzyme action through activation of RecA protein is proposed.     

The combined action of binase and bleomycin on human lung adenocarcinoma cells

Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations.     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS₄₀ and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1–5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS₄₀ and inhibits metastasis up to 50% in LLC and RLS₄₀ and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS₄₀-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.     

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.     

The cloning and expression of the RNAse structural gene of Bacillus intermedius]

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.     

The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.     

Genotoxicity of the preparation "binase" in tests on Salmonella: Ames test and ARA-test]

The present work deals with mutagenicity determination of enzyme sample "binase" (Bacillus intermedius ribonuclease) in microbial test-systems: Ames test and Ara-test. The weak mutagenic effect of "binase" high concentration was established in both tests by induction of forward Ara-mutations and Histidine-reverse mutations. A metabolic activation is seen to remove this effect.     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.