The present study compares two methods for the determination of fetal lung maturity: the novel in... more The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r= 0.77, p < 0.05, n= 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r= 0.94, p < 0.05, n= 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r= 0.75, p < 0.05, n= 29) and noncentrifuged (r= 0.63, p < 0.05, n= 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n= 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.
Molecular medicine (Cambridge, Mass.), Jan 4, 2015
Humanin and its derivatives are peptides known for their protective anti-apoptotic effects agains... more Humanin and its derivatives are peptides known for their protective anti-apoptotic effects against Alzheimer's disease. Herein, we identify a novel function of the humanin-derivative AGA(C8R)-HNG17-namely, protection against cellular necrosis. Necrosis is one of the main modes of cell death, which was until recently considered an unmoderated process. However, recent findings suggest the opposite. We have found that AGA(C8R)-HNG17 confers protection against necrosis in the neuronal cell lines PC-12 and NSC-34, where necrosis is induced in a glucose free medium by either chemohypoxia or by a shift from apoptosis to necrosis. Our studies in traumatic brain injury models in mice, where necrosis is the main mode of neuronal cell death, have shown that AGA(C8R)-HNG17 has a protective effect. This is demonstrated by a decrease in a neuronal severity score and by a reduction in brain edema as measured by MRI. An insight into the peptide's anti-necrotic mechanism was attained through...
Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtain... more Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in t...
Autophagy is involved in both the cell protective and the cell death process but its mechanism is... more Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;fished out&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.
Time-Resolved Laser Spectroscopy in Biochemistry II, 1990
ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was exa... more ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA&#39;s activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP&#39;s regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.
This article reports a novel approach for the evaluation of fetal lung maturity based on fluoresc... more This article reports a novel approach for the evaluation of fetal lung maturity based on fluorescence polarization (FP). The technique determines the intrinsic fluorescence polarization ratio (IFPR) of the amniotic fluid (AF). In vitro measurements of the IFPR indicate a clear dichotomy: high values for young pregnancies and low values for mature pregnancies. The new method has the potential to be a noninvasive procedure because the excitation of the AF and the collection of its fluorescence emission can be performed through the intact cervical amniotic membranes.
To detect and characterize membrane domains that have been proposed to exist in bacteria, two kin... more To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoylphosphatidylethanolamine (PY-PE) and 2-pyrenedecanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane. The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 ∞ ∞ ∞ ∞ C. This was ascribed to different local concentrations rather than to differences in mobility. The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E. coli and B. subtilis , in contrast to model membranes. In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order. Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes. It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE.
... Abraham H. Parola, Anita W. Rose, and Saul G. Cohen* Contribution ... and kinetic analy-sis w... more ... Abraham H. Parola, Anita W. Rose, and Saul G. Cohen* Contribution ... and kinetic analy-sis was made in accord with eq 2.2 However, light-absorbing transients prevented kinetic analysis in photoreduction of benzophenone by secondary and tertiary amines in ben-zene.2 ...
DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a centr... more DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3-O-(N-methylantraniloyl)-5-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.
, in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cell... more , in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.
Page 1. Photoreduction by Amines ... A. By Neat Amines and in Nonpolar Solvents B. Photoreduction... more Page 1. Photoreduction by Amines ... A. By Neat Amines and in Nonpolar Solvents B. Photoreduction in Aqueous Media C. Quenching of Phosphorescence of Benzophenones D. Reactions with Aromatic Amines E. Further Considerations 4-Benzoyl benzoate ...
Background: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One... more Background: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.
Title: Membrane Occupancy-Dependent Rejuvenation of DnaA Is Associated with Its Conformationally ... more Title: Membrane Occupancy-Dependent Rejuvenation of DnaA Is Associated with Its Conformationally Driven Oligomerization. Authors: Parola, Abraham H.; Aranovich, Alexander; Braier, Shani; Ansbacher, Esti; Rapoport, Hanna; Granek, Rony; Fishov, Itzhak. ...
The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was e... more The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was examined by two independent methods: 1) measurement of cell proliferation at 0.06 -0.7 mT (100, 60 and 50 Hz) using a colorimetric assay (MTT); 2) monitoring of specific activity of adenosine deaminase (ADA) at 0.3 and 0.7 mT, 60 Hz. Both increased cell proliferation and reduced ADA specific activity are associated with cell transformation. The MTT test showed an increase in cell proliferation of up to 64% after a 24 h exposure to SVMF at 100 Hz, 0.7 mT. Cell proliferation at constant frequency (100 Hz) depended on SVMF intensity. Cell proliferation at constant intensity (0.7 mT) increased with increasing field frequency. At 0.7 mT, 60 Hz cell proliferation increased by 31%, 28%, and 26% when measured by hemocytometry, 3 H-thymidine incorporation, and the MTT assay, respectively. ADA specific activity in CEF decreased by circa 48% on exposure to SVMF at 60 Hz, 0.3 mT for 24 h; only a statistically insignificant trend was seen at 0.7 mT, 60 Hz. Our findings showed that CEF cell proliferation and ADA specific activity were modified by SVMF. Both methods, independently, qualitatively detect a magnetic field effect.
The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethy1amino)napht... more The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethy1amino)naphthalene-? From the Departments of Chemistry (G.S. and A.H.P.) and Physics Abbreviations used: ADase, adenosine deaminase; 3'4-dansyladenosine, 3'4-[ 5-(dimethy1amino)naphthalene-1 -sulfonyl]adenosine; TLC, thin-layer chromatography; t-adenosine, 1,P-ethenoadenosine; e-ADP, 1,P-ethenoadenosine 5-diphosphate; t-NAD, nicotinamide 1 ,P-ethanoadenine dinucleotide; GPDH, glyceraldehyde-3-phosphate dehydrogenase; CPK, Corey-Pauling-Koltun.
DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once... more DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by ATP hydrolysis and ADP-ATP exchange. The latter can be promoted by binding to specific sequences on the chromosome and/or to acidic phospholipids in the membrane. We have previously shown that the transition into an active form (rejuvenation) is strongly co-operative with respect to DnaA membrane occupancy. Only at low membrane occupancy is DnaA reactivation efficiently catalysed by the acidic phospholipids. The present study was aimed at unravelling the molecular mechanism underlying the occupancy-dependent DnaA rejuvenation. We found that truncation of the DnaA N-terminal completely abolishes the co-operative transformation between the high and low occupancy states (I and II respectively) without affecting the membrane binding. The environmentally sensitive fluorophore specifically attached to the N-terminal cysteines of DnaA reported on occupancy-correlated changes in its vicinity. Cross-linking of DnaA with a short homobifunctional reagent revealed that state II of the protein on the membrane corresponds to a distinct oligomeric form of DnaA. The kinetic transition of DnaA on the membrane surface is described in the present study by a generalized 2D condensation phase transition model, confirming the existence of two states of DnaA on the membrane and pointing to the possibility that membrane protein density serves as an on-off switch in vivo. We conclude that the DnaA conformation attained at low surface density drives its N-terminal-mediated oligomerization, which is presumably a pre-requisite for facilitated nt exchange.
The present study compares two methods for the determination of fetal lung maturity: the novel in... more The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r= 0.77, p < 0.05, n= 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r= 0.94, p < 0.05, n= 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r= 0.75, p < 0.05, n= 29) and noncentrifuged (r= 0.63, p < 0.05, n= 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n= 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.
Molecular medicine (Cambridge, Mass.), Jan 4, 2015
Humanin and its derivatives are peptides known for their protective anti-apoptotic effects agains... more Humanin and its derivatives are peptides known for their protective anti-apoptotic effects against Alzheimer's disease. Herein, we identify a novel function of the humanin-derivative AGA(C8R)-HNG17-namely, protection against cellular necrosis. Necrosis is one of the main modes of cell death, which was until recently considered an unmoderated process. However, recent findings suggest the opposite. We have found that AGA(C8R)-HNG17 confers protection against necrosis in the neuronal cell lines PC-12 and NSC-34, where necrosis is induced in a glucose free medium by either chemohypoxia or by a shift from apoptosis to necrosis. Our studies in traumatic brain injury models in mice, where necrosis is the main mode of neuronal cell death, have shown that AGA(C8R)-HNG17 has a protective effect. This is demonstrated by a decrease in a neuronal severity score and by a reduction in brain edema as measured by MRI. An insight into the peptide's anti-necrotic mechanism was attained through...
Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtain... more Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in t...
Autophagy is involved in both the cell protective and the cell death process but its mechanism is... more Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;fished out&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.
Time-Resolved Laser Spectroscopy in Biochemistry II, 1990
ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was exa... more ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA&#39;s activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP&#39;s regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.
This article reports a novel approach for the evaluation of fetal lung maturity based on fluoresc... more This article reports a novel approach for the evaluation of fetal lung maturity based on fluorescence polarization (FP). The technique determines the intrinsic fluorescence polarization ratio (IFPR) of the amniotic fluid (AF). In vitro measurements of the IFPR indicate a clear dichotomy: high values for young pregnancies and low values for mature pregnancies. The new method has the potential to be a noninvasive procedure because the excitation of the AF and the collection of its fluorescence emission can be performed through the intact cervical amniotic membranes.
To detect and characterize membrane domains that have been proposed to exist in bacteria, two kin... more To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoylphosphatidylethanolamine (PY-PE) and 2-pyrenedecanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane. The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 ∞ ∞ ∞ ∞ C. This was ascribed to different local concentrations rather than to differences in mobility. The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E. coli and B. subtilis , in contrast to model membranes. In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order. Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes. It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE.
... Abraham H. Parola, Anita W. Rose, and Saul G. Cohen* Contribution ... and kinetic analy-sis w... more ... Abraham H. Parola, Anita W. Rose, and Saul G. Cohen* Contribution ... and kinetic analy-sis was made in accord with eq 2.2 However, light-absorbing transients prevented kinetic analysis in photoreduction of benzophenone by secondary and tertiary amines in ben-zene.2 ...
DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a centr... more DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3-O-(N-methylantraniloyl)-5-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.
, in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cell... more , in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.
Page 1. Photoreduction by Amines ... A. By Neat Amines and in Nonpolar Solvents B. Photoreduction... more Page 1. Photoreduction by Amines ... A. By Neat Amines and in Nonpolar Solvents B. Photoreduction in Aqueous Media C. Quenching of Phosphorescence of Benzophenones D. Reactions with Aromatic Amines E. Further Considerations 4-Benzoyl benzoate ...
Background: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One... more Background: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.
Title: Membrane Occupancy-Dependent Rejuvenation of DnaA Is Associated with Its Conformationally ... more Title: Membrane Occupancy-Dependent Rejuvenation of DnaA Is Associated with Its Conformationally Driven Oligomerization. Authors: Parola, Abraham H.; Aranovich, Alexander; Braier, Shani; Ansbacher, Esti; Rapoport, Hanna; Granek, Rony; Fishov, Itzhak. ...
The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was e... more The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was examined by two independent methods: 1) measurement of cell proliferation at 0.06 -0.7 mT (100, 60 and 50 Hz) using a colorimetric assay (MTT); 2) monitoring of specific activity of adenosine deaminase (ADA) at 0.3 and 0.7 mT, 60 Hz. Both increased cell proliferation and reduced ADA specific activity are associated with cell transformation. The MTT test showed an increase in cell proliferation of up to 64% after a 24 h exposure to SVMF at 100 Hz, 0.7 mT. Cell proliferation at constant frequency (100 Hz) depended on SVMF intensity. Cell proliferation at constant intensity (0.7 mT) increased with increasing field frequency. At 0.7 mT, 60 Hz cell proliferation increased by 31%, 28%, and 26% when measured by hemocytometry, 3 H-thymidine incorporation, and the MTT assay, respectively. ADA specific activity in CEF decreased by circa 48% on exposure to SVMF at 60 Hz, 0.3 mT for 24 h; only a statistically insignificant trend was seen at 0.7 mT, 60 Hz. Our findings showed that CEF cell proliferation and ADA specific activity were modified by SVMF. Both methods, independently, qualitatively detect a magnetic field effect.
The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethy1amino)napht... more The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethy1amino)naphthalene-? From the Departments of Chemistry (G.S. and A.H.P.) and Physics Abbreviations used: ADase, adenosine deaminase; 3'4-dansyladenosine, 3'4-[ 5-(dimethy1amino)naphthalene-1 -sulfonyl]adenosine; TLC, thin-layer chromatography; t-adenosine, 1,P-ethenoadenosine; e-ADP, 1,P-ethenoadenosine 5-diphosphate; t-NAD, nicotinamide 1 ,P-ethanoadenine dinucleotide; GPDH, glyceraldehyde-3-phosphate dehydrogenase; CPK, Corey-Pauling-Koltun.
DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once... more DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by ATP hydrolysis and ADP-ATP exchange. The latter can be promoted by binding to specific sequences on the chromosome and/or to acidic phospholipids in the membrane. We have previously shown that the transition into an active form (rejuvenation) is strongly co-operative with respect to DnaA membrane occupancy. Only at low membrane occupancy is DnaA reactivation efficiently catalysed by the acidic phospholipids. The present study was aimed at unravelling the molecular mechanism underlying the occupancy-dependent DnaA rejuvenation. We found that truncation of the DnaA N-terminal completely abolishes the co-operative transformation between the high and low occupancy states (I and II respectively) without affecting the membrane binding. The environmentally sensitive fluorophore specifically attached to the N-terminal cysteines of DnaA reported on occupancy-correlated changes in its vicinity. Cross-linking of DnaA with a short homobifunctional reagent revealed that state II of the protein on the membrane corresponds to a distinct oligomeric form of DnaA. The kinetic transition of DnaA on the membrane surface is described in the present study by a generalized 2D condensation phase transition model, confirming the existence of two states of DnaA on the membrane and pointing to the possibility that membrane protein density serves as an on-off switch in vivo. We conclude that the DnaA conformation attained at low surface density drives its N-terminal-mediated oligomerization, which is presumably a pre-requisite for facilitated nt exchange.
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Papers by Abraham Parola