Replication of phage phi 29 DNA in vitro: role of the viral protein p6 in initiation and elongation.

The phi 29 protein p6 stimulates the formation of the protein p3-dAMP initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 DNA polymerase p2 and phi 29 DNA-protein p3 complex, by decreasing about 5 fold the Km value for dATP. In addition, protein p6 stimulates elongation of the p3-dAMP initiation complex. Whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 DNA ends, the stimulation of elongation is higher with the right than with the left phi 29 DNA terminal fragment, suggesting DNA sequence specificity. The stimulation by protein p6 of the initiation and elongation steps of phi 29 DNA replication does not require the presence of the parental protein p3 at the phi 29 DNA ends. No effect of protein p6 was obtained on the elongation of the template-primer poly(dT)-(dA) 12-18 by the phi 29 DNA polymerase.     

Dipole potentials indicate restructuring of the membrane interface induced by gadolinium and beryllium ions

The dipole component of the membrane boundary potential, phi(d), is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Deltaphi(d), and apply it in studies of Be(2+) and Gd(3+) interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Deltaphi(d) is determined as the difference between the changes of the total boundary potential, phi(b), measured by the IFC method in planar lipid membranes and the surface potential, phi(s), determined from the electrophoretic mobility of liposomes. The Gouy-Chapman-Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be(2+) and Gd(3+) to PC membranes, and of Mg(2+) to PS membranes, the values of Deltaphi(b) and Deltaphi(s) are the same, indicative of no change of phi(d). Binding of Gd(3+) to PS-containing membranes induces changes of phi(d) of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Deltaphi(d) is negative (-30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be(2+) or Gd(3+) induces an increase of phi(d) of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd(3+) is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.     

Effect of NH4+ ions on phi 29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase.

Ammonium ions stimulated the formation of the phi diameter 29 protein p3-dAMP initiation complex by decreasing the Km value for dATP in a purified system containing the viral terminal protein p3, the viral DNA polymerase p2, and the phi 29 DNA-protein p3 complex as a template. In addition, NH4+ ions stimulated the amount of p3-dAMP complex elongation and increased by about twofold the rate of elongation. The stimulatory effect of NH4+ ions on in vitro phi 29 DNA replication is probably related to the formation of a stable complex between the terminal protein and the DNA polymerase, which was detected only in the presence of NH4+ ions.     

Peristaltic transport of a couple stress fluid in a uniform and non-uniform channels

The problem of peristaltic transport of a couple stress fluid in uniform and non-uniform two-dimensional channels has been investigated under zero Reynolds number with long wavelength approximation. Blood is represented by a couple stress fluid (a fluid which its particles size are taken into account, a special case of a non-Newtonian fluid). It is found that the pressure rise decreases as the couple stress fluid parameter gamma increases (i.e., small size fluid particle). So the pressure rise for a couple stress fluid (as a blood model) is greater than that for a Newtonian fluid. Also the pressure rise increases as the amplitude ratio phi increases for different values of gamma. Further, the pressure rise in the case of non-uniform geometry is found to be much smaller than the corresponding value in the case of uniform geometry. Finally, the maximum pressure rise when the mean flow rate over one period of the wave, Q = 0, increases as phi increases and gamma decreases.     

Electrodiffusion of ions approaching the mouth of a conducting membrane channel.

The movement of ions in the aqueous medium as they approach the mouth (radius a) of a conducting membrane channel is analyzed. Starting with the Nernst-Planck and Poisson equations, we derive a nonlinear integrodifferential equation for the electric potential, phi(r), a less than or equal to r less than infinity. The formulation allows deviations from charge neutrality and dependence of phi(r) on ion flux. A numerical solution is obtained by converting the equation to an integral equation that is solved by an iterative method for an assumed mouth potential, combined with a shooting method to adjust the mouth potential until the numerical solution agrees with an asymptotic expansion of the potential at r-a much greater than lambda (lambda = Debye length). Approximate analytic solutions are obtained by assuming charge neutrality (Läuger, 1976) and by linearizing. The linear approximation agrees with the exact solution under most physiological conditions, but the charge-neutrality solution is only valid for r much greater than lambda and thus cannot be used unless a much greater than lambda. Families of curves of ion flux vs. potential drop across the electrolyte, phi(infinity)-phi (a), and of permeant ion density at the channel mouth, n1(a), vs. flux are obtained for different values of a/lambda and S = a d phi/dr(a). If a much greater than lambda and S = O, the maximum flux (which is approached when n1(a)----0) is reduced by 50% compared to the value predicted by the charge-neutrality solution. Access resistance is shown to be a factor a/[2 (a + lambda)] times the published formula (Hille, 1968), which was derived without including deviations from charge neutrality and ion density gradients and hence does not apply when there is no counter-ion current. The results are applied to an idealized diffusion-limited channel with symmetric electrolytes. For S = O, the current/voltage curves saturate at a value dependent on a/lambda; for S greater than O, they increase linearly for large voltage.     

Assessing the role of HLA-linked and unlinked determinants of disease.

The relationship between increased risk in relatives over population prevalence (lambda R = KR/K) and probability of sharing zero marker alleles identical by descent (ibd) at a linked locus (such as HLA) by an affected relative pair is examined. For a model assuming a single disease-susceptibility locus or group of loci tightly linked to a marker locus, the relationship is remarkably simple and general. Namely, if phi R is the prior probability for the relative pair to share zero marker alleles identical by descent, then P (sharing 0 markers/both relatives are affected) is just phi R/lambda R. Alternatively, lambda AR, the increased risk over population prevalence to a relative R due to a disease locus tightly linked to marker locus A, equals the prior probability that the relative pair share zero A alleles ibd divided by the posterior probability that they share zero alleles ibd, given that they are both affected. For example, for affected sib pairs, P (sharing 0 markers/both sibs are affected) = .25/lambda S. This formula holds true for any number of alleles at the disease locus and for their frequencies, penetrances, and population prevalence. Similar formulas are derived for sharing one and two markers. Application of these formulas to several well-studied HLA-associated diseases yields the following results: For multiple sclerosis, insulin-dependent diabetes mellitus, and coeliac disease, a single-locus model of disease susceptibility is rejected, implying the existence of additional unlinked familial determinants. For all three diseases, the effect of the HLA-linked locus on familiality is minor: for multiple sclerosis, it accounts for only a 2.5-fold increased risk to sibs over the population prevalence, compared to an observed value of 20; for coeliac disease, it accounts for approximately a 5.25-fold increased risk to sibs, while the observed value is on the order of 60; for insulin-dependent diabetes mellitus, it accounts for a 3.42-fold increased risk in sibs, while the observed value is 15. In all cases, the secondary determinants must be outside the HLA region. For tuberculoid leprosy, an unlinked familial determinant is also implicated (increased risk to sibs due to HLA = 1.49; observed value = 2.38). For hemochromatosis and Hodgkin's disease, there is little evidence for HLA-unlinked familial determinants. With this formula, it is also possible to examine the hypothesis of pleiotropy versus linkage dis-equilibrium by comparing lambda AS with the increased risk to sibs due to the associated allele(s).(ABSTRACT TRUNCATED AT 400 WORDS)     

Behaviour of DNA-RNase A complex in the presence of formaldehyde]

A formaldehyde-produced fixation of defects caused by a despiralizing action of a protein was studied in the case of DNA-RNAase A complex. The concentration of the defects fixed was measured by kinetic formaldehyde method (KF-method). It was shown that following processes take place in the complex in the presence of formaldehyde: (a) fixation of defects; (b) unwinding of DNA; (c) inactivation of the protein. The rates of all these processes depend on the concentration of formaldehyde, phi. At formaldehyde concentrations above some critical value phic the protein is inactivated before the defects are fixed. At phi less than phic the protein inactivation proceeds more slowly than the fixation of defects; at sufficiently low formaldehyde concentration no inactivation of protein occurs practically during the fixation time (20 min). The number of new defects formed during the time of fixation is linear with the formaldehyde concentration in the region where no inactivation of the protein occurs. Therefore the initial concentration of defects can be determined through an extrapolation to zero concentration of formaldehyde. On the basis of the data obtained a method is proposed for the evaluation of the number of defects in DNA caused by the despiralizing action of proteins. A model is proposed describing the behaviour of the complexes of DNA with despiralizing proteins in the presence of formaldehyde.     

Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.     

Irreversible binding of phage phi X174 to cell-bound lipopolysaccharide receptors and release of virus-receptor complexes

At 37 degrees C, binding of phi X174 to the lipopolysaccharide receptors in the outer membrane of Escherichia coli C is followed by an irreversible ejection of its DNA. DNA ejection marks the beginning of the eclipse period in the infection cycle. Binding data with a phi X mutant Fcs70 at 15 degrees C, where the DNA ejection, or eclipse, rate is essentially zero, do not follow the law of mass action. This rules out a simple mechanism of reversible binding followed by irreversible DNA ejection. A more complex reaction model was devised to fit the data [Incardona, N. L. (1983) J. Theor. Biol. 105, 631-645]. It takes into account the fact that lipopolysaccharide-containing outer membrane fragments are continually released from infected E. coli cells, some of which have phi X bound to them. In this paper the model is shown to fit the binding data for wild-type virus at 15 degrees C and to account for the nonlinearity observed at 37 degrees C in the pseudo-first-order binding kinetics and first-order eclipse kinetics for both mutant and wild-type virus. This leads to the conclusion that phi X174 binding to cell-bound receptors is irreversible but binding to released receptors is reversible. The release of virus-receptor complexes from infected cells and the dissociation of these complexes were confirmed by electron microscopy. We propose that initially a single phi X174 vertex interacts reversibly with E. coli lipopolysaccharide but dissociation from the cell is prevented by the subsequent interaction of additional vertices with adjacent receptor molecules.     

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.     

Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability

Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.     

Rotation and protein-protein interactions of cytochrome P-450 in the inner membrane of adrenocortical mitochondria

Rotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011 beta) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-45011 beta. The observed value for the normalized time-independent anisotropy r(infinity)/r(0) and the average rotational relaxation time phi are r(infinity)/r(0) = 0.88 and phi = 233 microseconds when DOC is absent, and r(infinity)/r(0) = 0.65 and phi = 350 microseconds when DOC is present. Judging from the phi value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 A. A significantly high value of r(infinity)/r(0) implies co-existence immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55 degrees from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

An energy 'sources' and 'fractions' approach to the mechanical energy expenditure problem--IV. Criticism of the concept of 'energy transfers within and between links'

The methods of evaluation of mechanical energy economy during human movement based on the calculation of three different values of work (Wwb, Ww, Wn), corresponding to three hypothetical types of energy exchange, are subject to criticism. Only the value of work, Wwb, calculated under the assumption of energy transfers between links and energy transformations within links, can be useful as the lowest limit of mechanical energy expenditure (MEE) for the control (for the cases when the powers of the external sources are equal to zero). In the particular case when all of the joint powers Mi,i+1 (phi i+1 -phi i) have the same signs and all sources of external energy are absent, Wwb equals the MEE for the control.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Spin-lattice relaxation of coupled metal-radical spin-dimers in proteins: application to Fe(2+)-cofactor (Q(A)(-.), Q(B)(-.), phi(-.)) dimers in reaction centers from photosynthetic bacteria

The spin-lattice relaxation times (T(1)) for the reduced quinone acceptors Q(A)(-.) and Q(B)(-.), and the intermediate pheophytin acceptor phi(-.), were measured in native photosynthetic reaction centers (RC) containing a high spin Fe(2+) (S = 2) and in RCs in which Fe(2+) was replaced by diamagnetic Zn(2+). From these data, the contribution of the Fe(2+) to the spin-lattice relaxation of the cofactors was determined. To relate the spin-lattice relaxation rate to the spin-spin interaction between the Fe(2+) and the cofactors, we developed a spin-dimer model that takes into account the zero field splitting and the rhombicity of the Fe(2+) ion. The relaxation mechanism of the spin-dimer involves a two-phonon process that couples the fast relaxing Fe(2+) spin to the cofactor spin. The process is analogous to the one proposed by R. Orbach (Proc. R. Soc. A. (Lond.). 264:458-484) for rare earth ions. The spin-spin interactions are, in general, composed of exchange and dipolar contributions. For the spin dimers studied in this work the exchange interaction, J(o), is predominant. The values of J(o) for Q(A)(-.)Fe(2+), Q(B)(-.)Fe(2+), and phi(-.)Fe(2+) were determined to be (in kelvin) -0.58, -0.92, and -1.3 x 10(-3), respectively. The |J(o)| of the various cofactors (obtained in this work and those of others) could be fitted with the relation exp(-beta(J)d), where d is the distance between cofactor spins and beta(J) had a value of (0.66-0.86) A(-1). The relation between J(o) and the matrix element |V(ij)|(2) involved in electron transfer rates is discussed.     

Orientation of chlorophylls within chloroplasts as shown by optical and electrochromic properties of the photosynthetic membrane.

The effects on the optical properties of photosynthetic membranes caused by several types of chlorophyll differing in resonance frequency and in spatial disposition are theoretically analyzed. Using a method of moments and the linear dichroism spectrum of the lamellae, we evaluated the mean angle (phi) between the transition moment of each chlorophyll and the normal to the lamellae. We have confirmed that at about 695 nm the transition moment is in the plane of the lamellae, and outside it for chlorophyll b (phi approximately 48.6 degrees). By integrating over frequency the absorption variations affected by ionophores, we show that they may be ascribed to a Stark effect, and we analyze the dependence of this effect on the orientation of the chlorophylls. From this dependence and the degree of polarization of the Stark effect, we calculate the spatial fluctuations of the angle phi. The calculation shows that a definite value of phi corresponds to each resonance frequency of chlorophyl a found in vivo. This proves that the chlorophylls a are not oriented partly random. For chlorophylls b, on the other hand, phi may fluctuate by some 10 degrees about its mean value. The structural consequences of these results are discussed.     

Cooperative regulation of the Na+/H(+)-antiporter in Halobacterium halobium by delta pH and delta phi

The contributions of the transmembrane pH gradient (delta pH) and electrical potential (delta phi) to the delta mu H(+)-driven Na+ efflux (mediated by the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H(+)-antiporter) were investigated in membrane vesicles of Halobacterium halobium. Kinetic analysis in the dark revealed that two different Na(+)-binding sites are located asymmetrically across the membrane: One, accessible from the external medium, has a Kd (half-maximal stimulation of Na+ efflux) of about less than 50 mM, and the Na+ binding to the site is a prerequisite for the antiporter activation by delta mu H+. The other cytoplasmic site is the Na+ transport site. The Km for the cytoplasmic Na+ decreased as the delta pH increased, while the Vmax remained essentially constant in the presence of defined delta phi (140 mV). On the other hand, delta phi elevation above the gating potential (approximately 100 mV) increased the Vmax without changes in the Km in the presence of a fixed delta pH. It was also noted that the Km value in the absence of delta phi was completely different from and far higher than that observed in the presence of delta phi (greater than 100 mV), indicating the existence of two distinct conformations in the antiporter, resting and delta phi gated; the latter state may be reactive only to delta pH. On the basis of the present data and the previous data on the pH effect (N. Murakami and T. Konishi, 1989 Arch. Biochem. Biophys. 271, 515-523), a model for the delta pH-delta phi regulation of the antiporter activation is proposed.     

Inspection of human salivary alpha-amylase action by its transglycosylation action

The course of the action of human salivary alpha-amylase (HSA) on a substrate was examined taking advantage of its transglycosylation action. IG5 phi (IG-G-G-G-G-phi), IG4 phi (IG-G-G-G-phi), and GIG4 phi (G-IG-G-G-G-phi) were used as the substrates and p-nitrophenyl alpha-glucoside (GP, G-P) as the acceptor. HSA hydrolyzes IG5 phi, IG4 phi, and GIG4 phi to IG3 (IG-G-G) and G2 phi (G-G-phi), to IG3 and G phi (G-phi), and to GIG3 (G-IG-G-G) and G phi, respectively. In the presence of GP, a part of the glycon residues, IG3 and GIG3, were transferred to the acceptor to give IG4P (IG-G-G-G-P) and GIG4P (G-IG-G-G-G-P), respectively. Whenever the enzyme attacks the substrate, G phi or G2 phi is liberated in both transglycosylation and hydrolysis. The extent of transglycosylation can be, therefore, estimated from the molar ratio of the transfer product to the liberated aglycon, G phi or G2 phi. HPLC analysis of the reaction mixtures revealed that the value of IG4P/G phi in the digest of IG4 phi was nearly equal to that of GIG4P/G phi in the digest of GIG4 phi and these values were ten times larger than that of IG4P/G2 phi in the digest of IG5 phi. These data suggested that G phi residue would fall away from aglycon binding site more rapidly than G2 phi residue after the cleavage of the alpha-1,4-glycosidic linkage to offer GP more chance to attack to the activated glycon and also indicated that the space of the glycon binding site corresponds to three glucose residues.     

Quantum efficiencies of bacteriorhodopsin photochemical reactions.

Determination of quantum efficiencies of bacteriorhodopsin (bR) photoreactions is an essential step toward a full understanding of its light-driven proton-pumping mechanism. The bR molecules can be photoconverted into and from a K state, which is stable at 110 K. I measured the absorption spectra of pure bR, and the photoequilibrium states of bR and K generated with 420, 460, 500, 510, 520, 540, 560, 570, 580, 590, and 600 nm illumination at 110 K. The fraction of the K population in the photoequilibrium state, fk, is determined by AbR and AK the absorbances of the bR and K states at the excitation wavelengths, and also by phi 1 and phi 2, the quantum efficiencies for the bR to K and K to bR photoconversion: fK = phi 1 AbR/(phi 1AbR + phi 2Ak). By assuming that the ratio phi 1/phi 2 is the same at two different but close wavelengths, for example 570 and 580 nm, the value of phi 1/phi 2 at 570 and 580 nm was determined to be 0.55 +/- 0.02, and the spectrum of the K state was obtained with the peak absorbance at 607 nm. The values of phi 1/phi 2 at the other excitation wavelengths were then evaluated using the known K spectrum, and show almost no dependence on the excitation wavelength within the main band. The result phi 1/phi 2 = 0.55 +/- 0.02 disagrees with those of many other groups. The advantages of this method over others are its minimal assumptions and its straightforward procedure.