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V Brumfeld

Publications and source records attributed to V Brumfeld.

18 recordsLinked to original sources
PubMed1991

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures.

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm-1) to the lower wavelength (1457 cm-1) CH2 bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.

Animals↗
PubMed1991

Divalent cation-induced changes in conformation of protein kinase C.

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.

Animals↗
PubMed1990

Lipid dependence of surface conformations of protein kinase C.

The change of conformation of protein kinase C interacting with the surface of a mercury electrode directly from a solution or through a lipid monolayer was inferred from the number of cystine residues exposed and reduced on the electrode and from their reduction potentials. Soluble protein kinase C was estimated to have 5-6 disulfide bonds which could potentially react with the mercury electrode. Two major reduction peaks of cystine at different microenvironments within the protein molecule adsorbed to a mercury surface. They were observed in a.c. polarograms and cyclic voltamograms at two distinct potentials. The potential of these peaks became more negative as the pH of the solution increased, which was consistent with relaxation or decrease in alpha-helicity (ordered structure) of the protein as determined by circular dichroism (CD) estimations of secondary structure. The peak at the more positive potentials (-0.46 V relative to NAg/AgCl electrode at pH 7.4) tended to vanish upon cyclic reduction and reoxidation of the cystine, while the more negative peak (-0.62 V at pH 7.4) was enhanced. Addition of Mg2+ or Ca2+ had no significant effect on the potential but there was a reduction in their amplitude which appeared to affect the disappearance of these peaks upon pH adjustment. This suggests that the tertiary structure of the molecule is stabilized by Ca2+ and Mg2+, as substantiated by CD spectral analysis of secondary structures. Protein kinase C penetrated lipid monolayers to some extent. Addition of diacylglycerol or phorbol ester to the lipid monolayers facilitated this penetration. These compounds stabilized the protein surface conformation by destabilizing the monolayer at more positive potentials, resulting in an enhanced reduction peak at -0.42 V. This phenomenon was not significantly affected by Mg2+ or by Ca2+. The region of the protein kinase C (PKC) sequence which penetrated the monolayer contains cysteines and a primary amine(s), and may have homology to a region of phospholipase A2 which has been proposed as a phospholipid binding site for the two enzymes. Additionally, these polarographic studies suggest that PKC associates with and penetrates monolayers in a divalent cation-independent manner in agreement with our previous physical analyses of PKC interactions with lipid bilayers.

Adsorption↗
PubMed1990

Electric field dependence of alamethicin channels.

Circular dichroism (CD) of alamethicin embedded in vesicular membranes from outside, and its change, upon imposing Donnan potentials across the membrane, was measured. The changes in CD suggested a decrease in a helicity and increase in beta structure with the membrane potential positive inside and vice versa when the potential was positive on the outer side of the vesicles from where the alamethicin was inserted into the membrane. The Donnan potential was created by entrapping the polyacrylate (PA-) in the vesicles and changing the salt concentration outside or by adding different concentrations of PA- or polyethyleneimide (PEI+) at the outside of vesicles with 2 x 10(-5) M salt inside. The effect of the potential on the CD spectra and thus the alamethicin conformation is independent on the type of the polyelectrolyte employed for the Donnan potential generation.

Alamethicin↗
PubMed1990

Structural distinction between soluble and particulate protein kinase C species.

A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca2+/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more alpha-helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200-250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.

Chemical Phenomena↗
PubMed1990

Protein kinase C penetration into lipid bilayers.

Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.

Adenosine Triphosphate↗
PubMed1990

Ligand-induced conformational changes in cytosolic protein kinase C.

The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation.

Animals↗
PubMed1984

Interaction of influenza and parainfluenza viruses with polycations, organic oligocations and chromosome preparations.

As evaluated by light scattering at 90 degrees, natural organic oligocations such as putrescine, spermidine and spermine interfered with myxovirus aggregates which were induced by the histone H2A and strongly amplified by shaking during incubation. In contrast, the synthetic oligocation 1.7-diamino heptane itself aggregated the virus particles, its action being unmodified by adding polycationic H2A in abundance. When human chromosome preparations treated with protamine solution and shaked during incubation were covered with a stable polycationic molecular layer, the chromosomes had become unstainable by the Giemsa method even if the dye was used in excess. Nevertheless, the affinity of influenza virus particles for protamine was so high that they were able to dissociate the protamine molecules from the preformed complexes reconstituting the affinity of chromosome preparations to Giemsa stain. The virus-caused shift in the staining ability of chromosomes did not occur when bacterial suspension was added instead of the viral one. The model of oligocationic relaxation and of polycation condensation accounting for the modulatory effects of polyamines is discussed.

Cations↗
PubMedDate not supplied

Polycation-cell surface interactions and plasma membrane compartments in mammals. Interference of oligocation with polycationic condensation.

At lower concentrations, polyarginine, polylysine, protamine, histones H1, H2A, H2B and H3 cause lysis of human erythrocytes, whereas at higher concentrations inner histones are not hemolytic but induce only surface condensation and alterations in the cell-shape. Antibody coated erythrocytes treated with polyarginine result in ghost-like spheres having globular bodies 1 micron in diameter on the surface. Human fibroblasts and lymphocytes, and Ehrlich ascites cells treated with polyarginine also form surface globular bodies similar in size. Nucleate cell-polyarginine mixtures with lower polycation doses result in cytolysis, while higher polycation doses produce pyknosis of the cell surface accompanied by reorganization of a membrane-like structure. Changes in spectrofluorometric values result from 1,6-diphenyl-1,3, 5-hexatrien binding to cell lipids, match the plasma membrane alterations. Reciprocal shake incubation amplifies and/or conditions these polycation-induced alterations. The homogeneity of pyknotic surface bodies and the apparent polycationic membrane reorganization requiring oscillatory friction forces suggest the preexistence of a multizonal glycocalyx distribution corresponding to plasma membrane compartments. The possible role of this compartmentalization in receptor and membrane recycling, as well as the involvement of reversible catalytic-like polycation condensation in macromolecular changes are discussed.

Animals↗
PubMedDate not supplied

Characterization of an enzyme immunoassay by means of specific interaction affinities.

Antigen--antibody interactions involved in an enzyme immunoassay are characterized by their specific affinities. The use of a nonlinear least-squares approach of the response versus dose dependence allows the extrapolation of experimental data, as well as qualitative and/or quantitative assumptions about the interactions studied. As a practical test, the present technique has important advantages over common titration methods, by avoiding the fixation of a cut-off limit for positive and negative samples and by determining the affinity of the specific interaction for any dose required.

Animals↗
PubMedDate not supplied

Anti-influenza antibody assay by light scattering.

A physical method based on light scattering at 90 degrees was used to make evident the specific interaction between influenza virus and influenza antiserum. Purified virus put in contact with different dilutions of normal chicken serum exhibited a dose-effect dependence, which was significantly different from that corresponding to the virus-antiserum interaction. The maximum dilution of anti-influenza rabbit serum allowing the detection of the specific interaction between serum and infected allantoic fluid was then established. This value was considered to represent the "light scattering titer" of the antiserum.

Allantois↗
PubMedDate not supplied

Automation of laboratory instrumentation--recent achievements in the Stefan S. Nicolau Institute of Virology.

An automated system consisting of an SPF-500 spectrofluorometer interfaced to a Felix-M18 microcomputer was provided with a series of additional accessories benefitting from the inputs and outputs through the interface. An adequate set of programs for computer-controlled operations was developed. The system has allowed the performance of more than 15,000 absorption, emission and light scattering determinations. The role of interfacing to low cost computers in the automation of presently available laboratory instrumentation is discussed.

Academies and Institutes↗
PubMedDate not supplied

Light scattering from polycation--virus--oligocation mixtures.

The effect of a polycation (histone H2A) and of oligocations (spermine, spermidine and putrescine) on Sendai and influenza virus particles was investigated by light scattering measurements at 90 degrees and at various wavelengths. The method allows the study of the changes in the size and shape of virus particles in suspension.

Cations↗
PubMedDate not supplied

Polycation-induced alterations of the envelope surface of influenza and parainfluenza viruses.

Natural macromolecular polycations (protamine, histones H1, H2A, H2B and H3) caused the clustering of influenza and parainfluenza viruses into aggregates heterogeneous in size, as well as a decrease in their hemagglutinating capacity. Polycation-induced virus alterations consisted in surface pyknosis and an increase most efficient as regards the interaction with viruses.

Chemical Phenomena↗
PubMedDate not supplied

Felix-M18 microcomputer-controlled automation of spectrofluorometric measurements with applications in virology.

A system consisting of an Aminco SPF-500 spectrofluorometer interfaced to the Felix-M18 microcomputer was used in the automation of spectrofluorometric measurements with applications in virology. Programs, routines and subroutines in BASIC and in assembly language allow the operation of the spectrofluorometer and the processing of photometric data. The high efficiency of the system is illustrated by an application: the characterization of the fluorescence anisotropy value in polarized light of an influenza virus suspension preincubated with a fluorescent probe.

Computers↗