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PubMed1992

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.

Animals↗
PubMed1986

[A study on the mechanism of bile acid transport in the human placenta (the passive transport system of taurocholate across microvillous membrane)].

The uptake of taurocholate into microvillous membrane vesicles prepared from human normal full term placenta was studied using a rapid filtration technique. The taurocholate uptake into microvillous membrane vesicles was sensitive to extravesicular osmolarity, and preincubation with the taurocholate increased the uptake of taurocholate into the vesicles. These findings indicate that the uptake of taurocholate by microvillous membrane vesicles represents transport into vesicles. The uptake of taurocholate into vesicles was not dependent on sodium electrochemical gradient (extravesicular greater than intravesicular). But, this uptake was markedly increased when the intravesicular space was rendered electrically more positive by the use of lowly permeant anions or K+ diffusion potentials via valinomycin. These findings indicated that taurocholate was transported into microvillous membrane vesicles as anion. Cholic acid inhibited the uptake of taurocholate into vesicles, but taurine didn't inhibit this uptake. The initial rate of taurocholate transport exhibited saturation kinetics with respect to the taurocholate concentration; an apparent Km of 67 microM and Vmax of 0.30n mol/mg protein/20 sec were calculated. These results indicated that placental taurocholate transport was not active but passive (facilitated diffusion), and taurocholate was transported from fetus to mother via placenta because the blood concentration was higher in fetus than mother.

Bile Acids and Salts↗
PubMed2003

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line.

The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.

3-O-Methylglucose↗
PubMed2000

The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane.

We have investigated the mechanism responsible for the diffusive component of intestinal glucose absorption, the major route by which glucose is absorbed. In perfused rat jejunum in vivo, absorption was strongly inhibited by phloretin, an inhibitor of GLUT2. The GLUT2 level at the brush-border membrane increased some 2-fold when the luminal glucose concentration was changed from 0 to 100 mM. The phloretin-sensitive or diffusive component of absorption appeared superficially linear and consistent with simple diffusion, but was in fact carrier-mediated and co-operative (n=1.6, [G(1/2)]=56 mM; where [G(1/2)] is the glucose concentration at half V(max)) because of the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. Diffusive transport by paracellular flow was negligible. The phloretin-insensitive, SGLT1-mediated, component of glucose absorption showed simple saturation kinetics with [G(1/2)]=27 mM: the activation of protein kinase C (PKC) betaII, the isoenzyme of PKC that most probably controls GLUT2 trafficking [Helliwell, Richardson, Affleck and Kellett (2000) Biochem. J. 350, 149-154], also showed simple saturation kinetics, with [G(1/2)]=21 mM. We conclude that the principal route for glucose absorption is by GLUT2-mediated facilitated diffusion across the brush-border membrane, which is up to 3-fold greater than that by SGLT1; the magnitude of the diffusive component at any given glucose concentration correlates with the SGLT1-dependent activation of PKC betaII. The implications of these findings for the assimilation of sugars immediately after a meal are discussed.

Animals↗
PubMed1981

Transpleural diffusion of carbon dioxide.

Carbonic anhydrase in lung tissue might play a role in speeding the movement of CO2 between blood and alveolus. To test this hypothesis, we measured the transpleural diffusion rate of CO2 and compared it to that of oxygen, argon, and nitrogen, before and after inhibition of carbonic anhydrase activity with acetazolamide. Experiments were performed in exsanguinated dog lungs, which allowed study of CO2 dynamics in the absence of carbonic anhydrase activity from erythrocytes. The relative rate of movement of CO2 and the other gases into and out of the lung, agreed with that predicted solely on the basis of molecular weight and solubility. We conclude that there is no evidence for facilitated diffusion of CO2 across the pleural tissue.

Animals↗
PubMed1981

High- and low-affinity transport of D-glucose from blood to brain.

Measurements of the unidirectional blood-brain glucose flux in rat were incompatible with a single set of kinetic constants for transendothelial transport. At least two transfer mechanisms were present: a high-affinity, low-capacity system, and a low-affinity, high-capacity system. The low-affinity system did not represent passive diffusion because it distinguished between D- and L-glucose. The Tmax and Km for the high-affinity system were 0.16 mmol 100 g-1 min-1 and 1 mM; for the low-affinity system, approximately 5 mmol 100 g-1 min-1 and approximately 1 M. With these values, physiological glucose concentrations were not sufficient to saturate the low-affinity system. In normoglycemia, therefore, three independent pathways of glucose transport from blood to brain appear to exist: a high-affinity facilitated diffusion pathway of apparent permeability 235 X 10(-7) cm s-1, a specific but nonsaturable diffusion pathway of permeability 85 x 10(-7) cm s-1, and a nonspecific passive diffusion pathway of permeability 2 x 10(-7) cm s-1.

Animals↗
PubMed1983

Urea permeability of human red cells.

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.

Biological Transport↗
PubMed1992

Evidence for a saturable, energy-dependent and carrier-mediated uptake of oral antidiabetics into rat hepatocytes.

The hepatic uptake of two sulfonylureas, glisoxepide and glibenclamide, was investigated in isolated rat hepatocytes. Two transport processes were defined: passive physical diffusion and saturable carrier transport. For diffusion at pH 7.4 the permeability coefficients were 3.3 x 10(-6) cm/s for glisoxepide and 10.6 x 10(-6) cm/s for glibenclamide. Saturable uptake differed among the sulfonylureas. Glibenclamide uptake was neither energy- nor sodium-dependent and temperature dependence was linear. The apparent activation energy for saturable glibenclamide uptake was 15.2 kJ/mol and Q10 values for uptake between 7 and 37 degrees C were 1.17 +/- 0.12. Saturable glibenclamide uptake exhibited a Km = 3.1 microM and a Vmax = 416 pmol/mg cell protein per min. Thus glibenclamide uptake was defined kinetically as a facilitated diffusion process. Glisoxepide uptake revealed two Km values: Km1 = 2-3 microM and Vmax1 = 200 pmol/mg protein per min, and Km2 = 110 microM and Vmax2 = 1600 pmol/mg protein per min. Uptake at low and high substrate concentration was energy-dependent, sodium-dependent and was inhibited by ouabain. Temperature dependence increased markedly beyond 22 degrees C and the apparent activation energy was 59.7 kJ/mol at low Km1 glisoxepide concentrations and 60.3 kJ/mol at high Km2 concentrations. Whereas glisoxepide was slowly taken up into AS-30D hepatoma cells, glibenclamide was not. The hepatic uptake of glibenclamide was not inhibited by glisoxepide but glibenclamide inhibited glisoxepide uptake. The inhibition by glibenclamide was noncompetitive. Isolated hepatocytes accumulated the sulfonylureas markedly and metabolized both. The metabolized radioligands were slowly released into the incubation buffer. The results indicate that the hepatic uptake of the two sulfonylureas is by carrier-mediated transport. The uptake processes are, however, strikingly different, indicating heterogeneity of sulfonylurea transporters.

Administration, Oral↗
PubMed1986

Uptake of L-carnitine, D-carnitine and acetyl-L-carnitine by isolated guinea-pig enterocytes.

Uptake and metabolism of L-carnitine, D-carnitine and acetyl-L-carnitine were studied utilizing isolated guinea-pig enterocytes. Uptake of the D- and L-isomers of carnitine was temperature dependent. Uptake of L-[14C]carnitine by jejunal cells was sodium dependent since replacement by lithium, potassium or choline greatly reduced uptake. L- and D-carnitine developed intracellular to extracellular concentration gradients for total carnitine (free plus acetylated) of 2.7 and 1.4, respectively. However, acetylation of L-carnitine accounted almost entirely for the difference between uptake of L- and D-carnitine. About 60% of the intracellular label was acetyl-L-carnitine after 30 min, and the remainder was free L-carnitine. No other products were observed. D-Carnitine was not metabolized. Acetyl-L-carnitine was deacetylated during or immediately after uptake into intestinal cells and a portion of this newly formed intracellular free carnitine was apparently reacetylated. L-Carnitine and D-carnitine transport (after adjustment for metabolism and diffusion) were evaluated over a concentration range of 2-1000 microM. Km values of 6-7 microM and 5 microM, were estimated for L- and D-carnitine, respectively. Ileal-cell uptake was about half that found for jejunal cells, but the labeled intracellular acetylcarnitine-to-carnitine ratios were similar for both cell populations. Carnitine transport by guinea-pig enterocytes demonstrate characteristics of a carrier-mediated process since it was inhibited by D-carnitine and trimethylaminobutyrate, as well as being temperature and concentration dependent. The process appears to be facilitated diffusion rather than active transport since L-carnitine did not develop a significant concentration gradient, and was unaffected by ouabain or actinomycin A.

Acetylation↗
PubMed2001

Transport of aspartic acid, arginine, and tyrosine by the opportunistic protist Pneumocystis carinii.

In order to improve culture media and to discover potential drug targets, uptake of an acidic, a basic, and an aromatic amino acid were investigated. Current culture systems, axenic or co-cultivation with mammalian cells, do not provide either the quantity or quality of cells needed for biochemical studies of this organism. Insight into nutrient acquisition can be expected to lead to improved culture media and improved culture growth. Aspartic acid uptake was directly related to substrate concentration, Q(10) was 1.10 at pH 7.4. Hence the organism acquired this acidic amino acid by simple diffusion. Uptake of the basic amino acid arginine and the aromatic amino acid tyrosine exhibited saturation kinetics consistent with carrier-mediated mechanisms. Kinetic parameters indicated two carriers (K(m)=22.8+/-2.5 microM and K(m)=3.6+/-0.3 mM) for arginine and a single carrier for tyrosine (K(m)=284+/-23 microM). The effects of other L-amino acids showed that the tyrosine carrier was distinct from the arginine carriers. Tyrosine and arginine transport were independent of sodium and potassium ions, and did not appear to require energy from ATP or a proton motive force. Thus facilitated diffusion was identified as the mechanism of uptake. After 30 min of incubation, these amino acids were incorporated into total lipids and the sedimentable material following lipid extraction; more than 90% was in the cellular soluble fraction.

Arginine↗
PubMed1990

Effect of herpes simplex virus type 1 infection on nucleoside transport in HeLa S3 cells.

The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.

Affinity Labels↗
PubMed1976

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.

Animals↗
PubMed1981

Hexose transport in normal and SV40-transformed human endothelial cells in culture.

The mechanism of glucose entry into human vascular endothelial cells was studied in monolayer cultures of normal (primary) and virally (SV40) transformed umbilical vein endothelium. Radioisotopic uptake studies with the glucose analogues 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, and the nonmetabolizable stereoisomer L-glucose, indicated the presence of a saturable, stereospecific hexose carrier mechanism in both cell types. In other experiments with D-glucose and 3-O-methyl-D-glucose, the phenomenon of countertransport was demonstrable. Hexose transport was not affected by KCN, dinitrophenol, or ouabain, but was inhibited by phloretin and phlorizin in a pattern consistent with facilitated diffusion. Kinetic constants were obtained for both 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Similar Km values (range, 3.3-4.7 mM) were noted with normal and transformed cells, whereas the apparent Vmax was 0.56 nmol/microliter cytosol/minute for primary cells and 1.7-2.5 nmol/mu cytosol/minute for transformed cells. Under standard culture conditions, as well as following 18 hours of serum deprivation, insulin at concentrations up to 10(-5) M did not appear to influence hexose uptake in either cell type. Metabolism of 14C(U)-D-glucose to 14CO2 also was not stimulated by insulin. The presence of an insulin-insensitive, facilitated transport system for glucose in vascular endothelium has relevance for glucose metabolism in this tissue, and potentially for the association of certain vascular diseases (e.g., diabetic microangiopathy, atherosclerosis) with altered glucose homeostasis.

Biological Transport↗
PubMed2006

Modeling the role of incisures in vertebrate phototransduction.

Phototransduction is mediated by a G-protein-coupled receptor-mediated cascade, activated by light and localized to rod outer segment (ROS) disk membranes, which, in turn, drives a diffusion process of the second messengers cGMP and Ca2+ in the ROS cytosol. This process is hindered by disks-which, however, bear physical cracks, known as incisures, believed to favor the longitudinal diffusion of cGMP and Ca2+. This article is aimed at highlighting the biophysical functional role and significance of incisures, and their effect on the local and global response of the photocurrent. Previous work on this topic regarded the ROS as well stirred in the radial variables, lumped the diffusion mechanism on the longitudinal axis of the ROS, and replaced the cytosolic diffusion coefficients by effective ones, accounting for incisures through their total patent area only. The fully spatially resolved model recently published by our group is a natural tool to take into account other significant details of incisures, including their geometry and distribution. Using mathematical theories of homogenization and concentrated capacity, it is shown here that the complex diffusion process undergone by the second messengers cGMP and Ca2+ in the ROS bearing incisures can be modeled by a family of two-dimensional diffusion processes on the ROS cross sections, glued together by other two-dimensional diffusion processes, accounting for diffusion in the ROS outer shell and in the bladelike regions comprised by the stack of incisures. Based on this mathematical model, a code has been written, capable of incorporating an arbitrary number of incisures and activation sites, with any given arbitrary distribution within the ROS. The code is aimed at being an operational tool to perform numerical experiments of phototransduction, in rods with incisures of different geometry and structure, under a wide spectrum of operating conditions. The simulation results show that incisures have a dual biophysical function. On the one hand, since incisures line up from disk to disk, they create vertical cytoplasmic channels crossing the disks, thus facilitating diffusion of second messengers; on the other hand, at least in those species bearing multiple incisures, they divide the disks into lobes like the petals of a flower, thus confining the diffusion of activated phosphodiesterase and localizing the photon response. Accordingly, not only the total area of incisures, but their geometrical shape and distribution as well, significantly influence the global photoresponse.

Animals↗
PubMed2004

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB.

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.

Acetates↗
PubMed1996

Acid/base transport in a model of the proximal tubule brush border: impact of carbonic anhydrase.

A mathematical model of the brush border of the proximal tubule (T. A. Krahn, P. S. Aronson, and A. M. Weinstein. Bull. Math. Biol, 56: 459-490, 1994) has been extended by the inclusion of CO2 and H2CO3 as diffusible species and by the inclusion of finite rate constants for the hydration of CO2. This permits the simulation of carbonic anhydrase (CA) activity and its inhibition. We confirm the result of our previous study, which is that, in the presence of CA, the unstirred layer has only a modest effect on the observed formic acid permeability. CA inhibition results in disequilibrium pH gradients, and the effect of these gradients on formic acid permeability depends on the presence of other membrane transport proteins. We also examined the impact of CA activity on the flux of total CO2 through the brush border. Under physiological conditions, CA inhibition depressed NaHCO3 reabsorption through the brush border by interfering with the HCO3(-)-facilitated diffusion of CO2. However, the determination of brush-border CO2 permeability, using an imposed CO2 gradient, was relatively uninfluenced by CA activity. Finally, we inserted a kinetic representation of the Na+/H+ exchanger into the brush-border model. Even when luminal and cytosolic diffusion coefficients were increased 1,000-fold, there was no effect on brush-border Na+ flux. This suggests that variations in the unstirred layer cannot be responsible for the flow dependence of Na+ reabsorption.

Absorption↗
PubMed2002

Concerted binding and bending of DNA by Escherichia coli integration host factor.

Integration host factor (IHF) is a heterodimeric Escherichia coli protein that plays essential roles in a variety of cellular processes including site-specific recombination, transcription, and DNA replication. The IHF-DNA interface extends over three helical turns and includes sequential minor groove contacts that present strong, sequence specific protection patterns against hydroxyl radical cleavage. Synchrotron X-ray footprinting has been used to follow the kinetics of formation of DNA-protein contacts in the IHF-DNA complex with single base-pair spatial, and millisecond time, resolution. The three sites of IHF protection on the DNA develop with similar time-dependence, indicating that sequence specific binding and bending occur concertedly. Two distinct phases are observed in the association process. The first "burst" phase is characterized by a rate that is greater than diffusion limited (>10(10) s(-1) M(-1)) and the second phase is on the order of diffusion controlled (approximately 10(8) M(-1) s(-1)). The overall kinetics of association become faster with increasing IHF concentration showing that complex formation is second-order with protein. The rate of association is maximal between 100 and 200 mM KCl decreasing at higher and lower concentrations. The rate of IHF dissociation from site-specifically bound DNA increases monotonically as KCl concentration is increased. The dissociation progress curves are biphasic with the amplitude of the first phase dependent upon competitor DNA concentration. These results are the first analysis by synchrotron footprinting of the fast kinetics of a protein-DNA interaction and suggest that IHF binds its specific site through a multiple-step mechanism in which the first step is facilitated diffusion along the length of the duplex followed by subsequent binding and bending of the DNA in a concerted manner.

Bacterial Proteins↗
PubMed1994

A Re-Evaluation of the Role of the Infected Cell in the Control of O2 Diffusion in Legume Nodules.

Two different simulation models were constructed to describe O2 diffusion into the bacteria-infected cells of legume nodules: one based on a central zone of uniform spherical cells and the other on a central zone of packed, uniform cubical cells with air spaces along the edges. The cubical model more closely approximated the geometry and gas diffusion characteristics of infected cells than did the spherical model. The models relied on set values for the innermost O2 concentration in the infected cell (1-20 nM) and predicted values for the free O2 and oxygenated leghemoglobin gradients toward the cell:space interface. The cubical model but not the spherical model predicted saturation of leghemoglobin (Lb) oxygenation at or within a few micrometers of the gas-filled intercellular space and predicted that the space concentration could be as high as 1.3% O2 when the fractional oxygenation of Lb and respiration rate within the infected cell were typical of that which has been measured in vivo. In the model, the higher the space O2 concentration, the greater the saturation of Lb by O2 and the greater the collapse of Lb-facilitated diffusion near the cell:space interface. This was predicted to result in a greater resistance to O2 diffusion from the space to the bacteroids, thereby providing an intrinsic, homeostatic mechanism for controlling the rate of O2 influx into infected cells. Changes in the physiological features of the simulated cubical infected cell, such as the proportion of the cell as cytosol, the surface area of the cell exposed to a space, the maximum rate of cellular respiration, or the concentration of Lb in the cytoplasm, significantly altered the extent to which the infected cell would be able to regulate its diffusive resistance. These results demonstrate the possibility of a Lb-based mechanism for controlling the O2 concentration within the infected cells. If such a mechanism exists in legume nodules, it would give the infected cell an ability to exercise fine control over its internal environment, a process that could complement a physical diffusion barrier that may exist in the inner cortex or elsewhere in the nodule and provide coarse control over O2 diffusion.

Journal Article↗