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PubMed2004

Modification of surface crystal formation on mother crystal by additive molecules.

Additive molecules and excess reactant species (NaOH and NH4F) in NaF crystallization were used to control surface crystal formation on a mother crystal. When the surface integration was inhibited or bulk diffusion facilitated, more surface crystals were found on the mother crystal due to an increase in the interfacial supersaturation on the crystal. The inclusion of KCl as an additive and increases in the reactant concentration and agitation speed resulted in a higher population of surface crystals on the mother crystal, because surface integration was inhibited. However, surface crystal formation was suppressed by the polymer gelatin due to retardation of the bulk diffusion by the gelatin layer formed around the crystal. In addition, surface crystal formation significantly changed according to the feeding mode, and the excess species of NaOH originating from the NH4F feeding mode were more effective in facilitating surface crystal formation than the excess species of NH4F coming from the NaOH feeding mode due to the smaller ionic radii of NaOH than of NH4F.

Journal Article↗
PubMed2005

Functional challenge affects aquaporin mRNA abundance in mouse blastocysts.

The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the trophoblast cells. These are believed to be involved in facilitating the accumulation of fluid into the blastocyst cavity. We have investigated the ability of mouse embryos to regulate AQP gene expression in response to different treatments expected to affect the passage of water across the trophoblast cells using real-time PCR. In the first experiment 8-cell embryos were allowed to develop to blastocysts in media from 300 to 400 mOsm. Blastocyst formation was unaffected by media made hyperosmolar by glycerol, whereas blastocyst formation was significantly reduced in sucrose-based 350 and 400 mOsm media. AQP 8 mRNA levels were reduced when embryos were cultured in glycerol-based hyperosmolar media. The mRNA levels of AQP 3, 7, 9, and 11 were not significantly affected by hyperosmolar media. In the second experiment blastocysts were punctured (0 hr) and allowed to re-expand. AQP mRNA levels were examined after 2, 6, and 10 hr. Compared to control embryos, the expression of AQP 3, 7, and 9 were upregulated after 2 hr. Upregulation was sustained only for AQP 9 and this was sustained up to 6 and 10 hr after puncture. In the third experiment we compared expression of AQPs between in vitro cultured and in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations.

Actins↗
PubMed2004

Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk.

Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits gamma-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.

Animals↗
PubMed2005

Roles of aquaporin-3 water channels in volume-regulatory water flow in a human epithelial cell line.

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl(2)) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.

Aquaporin 3↗
PubMed1997

Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy.

We recently demonstrated that pentosidine, an advanced glycation end product, accumulates markedly as albumin-linked form (Palb) and in free-form (Pfree) in the plasma of patients with end-stage renal failure. The present study was undertaken to examine the clearance of Palb and Pfree by different modalities of renal replacement therapy, that is, hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD), and renal transplantation. HD cleared Pfree (9.4 +/- 4.3 nmol/kg/HD) but not Palb, by diffusion but not by membrane adsorption, whereas CAPD cleared both Palb (4.03 +/- 2.01 nmol/kg/day) and Pfree (2.43 +/- 1.24 nmol/kg/day). Plasma total pentosidine levels were significantly (P < 0.05) lower in CAPD (0.97 +/- 0.41 nmol/ml) than in HD (1.19 +/- 0.41 nmol/ml), as the result of a lower serum albumin level in the former patients. Indeed, Palb expressed per mg albumin was virtually identical in HD and CAPD. By contrast, Pfree was significantly lower in CAPD than in HD. Palb levels were significantly correlated with plasma Pfree levels in both HD and CAPD patients, but not in the CAPD dialysate. Pentosidine transport across the peritoneum occurs mainly by diffusion, both as Palb and Pfree. Interestingly, peritoneal Palb clearance (0.17 +/- 0.07 ml/min) significantly (P < 0.00001) exceeded albumin clearance (0.11 +/- 0.05 ml/min). Palb levels being significantly higher (P < 0.0005) in the peritoneal fluid (36.28 +/- 18.55 pmol/mg) than in the serum (27.12 +/- 11.71 pmol/mg), thus raises the possibility of a facilitated diffusion of Palb or an active transport mechanism for protein-linked pentosidine into the peritoneal cavity. After renal transplantation, plasma Pfree fell rapidly, remained barely detectable after one month, and returned to normal at six months. By contrast, Palb fell more slowly and remained significantly above normal at six months, but returned eventually to normal levels. These findings demonstrate that: (1) both HD and CAPD remove Pfree; (2) the peritoneal clearance of Palb, might contribute to the lower level of plasma pentosidine in CAPD than in HD patients; and (3) renal transplantation is the best therapeutic modality to normalize both Palb and Pfree levels.

Adult↗
PubMed2001

Modeling advection and diffusion of oxygen in complex vascular networks.

A realistic geometric model for the three-dimensional capillary network geometry is used as a framework for studying the transport and consumption of oxygen in cardiac tissue. The nontree-like capillary network conforms to the available morphometric statistics and is supplied by a single arterial source and drains into a pair of venular sinks. We explore steady-state oxygen transport and consumption in the tissue using a mathematical model which accounts for advection in the vascular network, nonlinear binding of dissolved oxygen to hemoglobin and myoglobin, passive diffusion of freely dissolved and protein-bound oxygen, and Michaelis-Menten consumption in the parenchymal tissue. The advection velocity field is found by solving the hemodynamic problem for flow throughout the network. The resulting system is described by a set of coupled nonlinear elliptic equations, which are solved using a finite-difference numerical approximation. We find that coupled advection and diffusion in the three-dimensional system enhance the dispersion of oxygen in the tissue compared to the predictions of simplified axially distributed models, and that no "lethal corner," or oxygen-deprived region occurs for physiologically reasonable values for flow and consumption. Concentrations of 0.5-1.0 mM myoglobin facilitate the transport of oxygen and thereby protect the tissue from hypoxia at levels near its P50, that is, when local oxygen consumption rates are close to those of delivery by flow and myoglobin-facilitated diffusion, a fairly narrow range.

Animals↗
PubMed1981

Diffusion kinetics of tritiated uridine into HeLa cells previously exposed to hyperthermia.

Hyperthermic treatment of HeLa cells at 42 degrees C for 60 min depressed the specific activity of these cells when incubated with 3H-uridine both during and post heating compared to cells maintained at 37 degrees C. These changes were unlikely to arise from increased leakage from the cells and may partially be attributed to membrane damage influencing facilitated diffusion. Diffusion kinetic data for incorporation of the radiolabel into the T.C.A. soluble and T.C.A. insoluble fractions of HeLa cells indicated that a significant depression of Vmax and a significant elevation of Km for incorporation of 3H UdR into RNA may possibly result from an isotope dilution effect attributed to degrading pre-ribosomal RNA under the effect of hyperthermia.

Diffusion↗
PubMed1986

Role of the alveolar macrophage in the induction of pulmonary phospholipidosis by chlorphentermine. II. Drug uptake into cells in vitro.

This study was conducted to further assess the role of the alveolar macrophage in the induction of pulmonary phospholipidosis by the cationic amphiphilic drug, chlorphentermine (CP). Alveolar macrophages were collected from normal rats by pulmonary lavage, allowed to attach to glass cover slips and incubated with CP in vitro at 37 degrees C. The uptake of CP was measured using [14C]CP. Uptake is rapid, reaching equilibrium by 2 min resulting in the concentration of CP within the cells. The process is temperature-dependent being depressed markedly at 2 degrees C. Two components of uptake were identified. Below 0.2 mM CP, a carrier-mediated mechanism and diffusion are involved whereas, at concentrations above 0.2 mM, the carrier is saturated and diffusion predominates, with the intracellular binding of CP to membranes probably responsible for the striking sequestration. The carrier-mediated component obeys Michaelis-Menten kinetics, does not appear to require Na+ and is not affected by metabolic inhibitors. This is consistent with the concept that the process occurs by facilitated diffusion. Metabolism of CP does not play a role in the accumulation of the drug. The transport system is different from those involved in glucose, nucleoside or amino acid uptake. Total initial uptake was inhibited by the three cationic amphilic drugs tested, iprindole, chlorcyclizine and imipramine indicating that cationic amphiphilic drugs may share a common uptake system.

Animals↗
PubMed2004

Drug transfer and metabolism by the human placenta.

The major function of the placenta is to transfer nutrients and oxygen from the mother to the foetus and to assist in the removal of waste products from the foetus to the mother. In addition, it plays an important role in the synthesis of hormones, peptides and steroids that are vital for a successful pregnancy. The placenta provides a link between the circulations of two distinct individuals but also acts as a barrier to protect the foetus from xenobiotics in the maternal blood. However, the impression that the placenta forms an impenetrable obstacle against most drugs is now widely regarded as false. It has been shown that that nearly all drugs that are administered during pregnancy will enter, to some degree, the circulation of the foetus via passive diffusion. In addition, some drugs are pumped across the placenta by various active transporters located on both the fetal and maternal side of the trophoblast layer. It is only in recent years that the impact of active transporters such as P-glycoprotein on the disposition of drugs has been demonstrated. Facilitated diffusion appears to be a minor transfer mechanism for some drugs, and pinocytosis and phagocytosis are considered too slow to have any significant effect on fetal drug concentrations. The extent to which drugs cross the placenta is also modulated by the actions of placental phase I and II drug-metabolising enzymes, which are present at levels that fluctuate throughout gestation. Cytochrome P450 (CYP) enzymes in particular have been well characterised in the placenta at the level of mRNA, protein, and enzyme activity. CYP1A1, 2E1, 3A4, 3A5, 3A7 and 4B1 have been detected in the term placenta. While much less is known about phase II enzymes in the placenta, some enzymes, in particular uridine diphosphate glucuronosyltransferases, have been detected and shown to have specific activity towards marker substrates, suggesting a significant role of this enzyme in placental drug detoxification. The increasing experimental data on placental drug transfer has enabled clinicians to make better informed decisions about which drugs significantly cross the placenta and develop dosage regimens that minimise fetal exposure to potentially toxic concentrations. Indeed, the foetus has now become the object of intended drug treatment. Extensive research on the placental transfer of drugs such as digoxin and zidovudine has assisted with the safe treatment of the foetus with these drugs in utero. Improved knowledge regarding transplacental drug transfer and metabolism will result in further expansion of pharmacological treatment of fetal conditions.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters.

A cDNA was isolated from rat small intestine by expression cloning which encodes a novel Na+-independent transporter for aromatic amino acids. When expressed in Xenopus oocytes, the encoded protein designated as TAT1 (T-type amino acid transporter 1) exhibited Na+-independent and low-affinity transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine (Km values: approximately 5 mm), consistent with the properties of classical amino acid transport system T. TAT1 accepted some variations of aromatic side chains because it interacted with amino acid-related compounds such as l-DOPA and 3-O-methyl-DOPA. Because TAT1 accepted N-methyl- and N-acetyl-derivatives of aromatic amino acids but did not accept their methylesters, it is proposed that TAT1 recognizes amino acid substrates as anions. Consistent with this, TAT1 exhibited sequence similarity (approximately 30% identity at the amino acid level) to H+/monocarboxylate transporters. Distinct from H+/monocarboxylate transporters, however, TAT1 was not coupled with the H+ transport but it mediated an electroneutral facilitated diffusion. TAT1 mRNA was strongly expressed in intestine, placenta, and liver. In rat small intestine TAT1 immunoreactivity was detected in the basolateral membrane of the epithelial cells suggesting its role in the transepithelial transport of aromatic amino acids. The identification of the amino acid transporter with distinct structural and functional characteristics will not only facilitate the expansion of amino acid transporter families but also provide new insights into the mechanisms of substrate recognition of organic solute transporters.

Amino Acid Sequence↗
PubMed1978

Stopped-flow spectrophotometric assay of glycerol permeation in Escherichia coli: applicability and limitations.

The passive permeation and facilitated diffusion of glycerol in various strains of Escherichia coli have been studied by stopped-flow spectrophotometry. Contrary to the prediction for glycerol entry by simple diffusion, the reciprocal relaxation time (1/tau, s-1) for the passive permeation of glycerol in cells grown in the presence of glucose was not constant but decreased as the glycerol concentration increased above 100 mM. This anomaly was not due to refractive index differences or to the presence of residual levels of the glycerol facilitator protein in non-induced cells. Although reciprocal relaxation times for glycerol-induced E. coli exhibited the expected elevation relative to non-induced cells, a similar anomalous decrease 1/tar (s-1) with increasing glycerol concentration was observed. In addition, at early times after suspension in dilute buffer, the 1/tau (s-1) values obtained for induced or non-induced E. coli swelling in glycerol were considerably greater than for organisms incubated in dilute buffer for longer times. We concluded that either this spectrophotometric technique was not monitoring solely the permeation of glycerol into E. coli, or concentrations of glycerol above 100 mM significantly perturbed the structure of the E. coli cell envelope.

Cell Count↗
PubMed1992

Energetics of arginine and lysine transport by whole cells and membrane vesicles of strain SR, a monensin-sensitive ruminal bacterium.

Strain SR, a monensin-sensitive, ammonia-producing ruminal bacterium, grew rapidly on arginine and lysine, but only if sodium was present. Arginine transport could be driven by either an electrical potential or a chemical gradient of sodium. Arginine was converted to ornithine, and it appeared that ornithine efflux created a sodium gradient which in turn drove arginine transport. There was a linear decline in arginine transport as pH was decreased from 7.5 to 5.5, and the cells did not grow at a pH less than 6.0. The Eadie-Hofstee plot was biphasic, and arginine could also be taken by a high-capacity diffusion mechanism. Because arginine was a strong inhibitor of lysine transport and lysine was a weak inhibitor of arginine transport, it appeared that both lysine and arginine were taken up by an arginine-lysine carrier which had a preference for arginine. The rate of lysine fermentation was always proportional to the extracellular lysine concentration, and facilitated diffusion was the dominant mechanism of lysine transport. When SR was grown in continuous culture on arginine or lysine, the theoretical maximal growth yield was similar (13 g of cells per mol of ATP), but the apparent maintenance energy requirement for arginine was greater than lysine (9.4 versus 4.4 mmol of ATP per g of cells per h). On the basis of differences in yield and maintenance energy, it appeared that active arginine transport accounted for approximately 40% of the total ATP.

Animals↗
PubMedDate not supplied

Differentiation of HL-60 cells by dimethylsulfoxide activates a Na(+)-dependent nucleoside transport system.

Uridine transport in undifferentiated HL-60 cells occurs primarily by facilitated diffusion, but a limited Na(+)-dependent process can be demonstrated (Km = 44 +/- 4.4 microM, Vmax = 0.13 +/- 0.01 microM/s). This latter transport system was inhibited by adenosine and inosine (Ki = 110 and 260 microM, respectively), whereas guanosine and thymidine were less effective (Ki = 1600 and 1200 microM, respectively). Dimethylsulfoxide (DMSO) caused a concentration-dependent decrease in facilitated uridine transport. This change was attributable to a decrease in the number of transporter molecules as determined by the binding of [3H]nitrobenzylthioinosine to cell membranes. Moreover, the Na(+)-dependent transport of uridine was enhanced by DMSO at a concentration of the polar solvent as low as 0.4%. When HL-60 cells were exposed to 1.0% DMSO, a marked increase in Na(+)-dependent uridine transport occurred within 72 hr, a time preceding maximum granulocytic differentiation. This change was attributable to an increase in transport affinity (Km = 1.54 +/- 0.65 microM), with no change in Vmax (Vmax = 0.13 +/- 0.02 microM/s). The consequence of these changes was the generation of a 3- to 4-fold increase in the intracellular concentration of uridine relative to the medium at a physiological concentration of 5 microM uridine. Similar increases in transport affinity were observed for adenosine, inosine, guanosine and thymidine in DMSO-differentiated HL-60 cells (Km values of 2 to 5 microM). These results complement our previous studies with phorbol 12-myristate 13-acetate, in which differentiation to a monocytic phenotype was also associated with enhanced Na(+)-dependent nucleoside transport.

Biological Transport↗
PubMed2003

Pulsatile urea excretion in the gulf toadfish: mechanisms and controls.

Opsanus beta expresses a full complement of ornithine-urea cycle (OUC) enzymes and is facultatively ureotelic, reducing ammonia-N excretion and maintaining urea-N excretion under conditions of crowding/confinement. The switch to ureotelism is keyed by a modest rise in cortisol associated with a substantial increase in cytosolic glutamine synthetase for trapping of ammonia-N and an upregulation of the capacity of the mitochondrial OUC to use glutamine-N. The entire day's urea-N production is excreted in 1 or 2 short-lasting pulses, which occur exclusively through the gills. The pulse event is not triggered by an internal urea-N threshold, is not due to pulsatile urea-N production, but reflects pulsatile activation of a specific branchial excretion mechanism that rapidly clears urea-N from the body fluids. A bidirectional facilitated diffusion transporter, with pharmacological similarity to the UT-A type transporters of the mammalian kidney, is activated in the gills, associated with an increased trafficking of dense-cored vesicles in the pavement cells. An 1814 kB cDNA ('tUT') coding for a 475-amino acid protein with approximately 62% homology to mammalian UT-A's has been cloned and facilitates phloretin-sensitive urea transport when expressed in Xenopus oocytes. tUT occurs only in gill tissue, but tUT mRNA levels do not change over the pulse cycle, suggesting that tUT regulation occurs at a level beyond mRNA. Circulating cortisol levels consistently decline prior to a pulse event and rise thereafter. When cortisol is experimentally clamped at high levels, natural pulse events are suppressed in size but not in frequency, an effect mediated through glucocorticoid receptors. The cortisol decline appears to be permissive, rather than the actual trigger of the pulse event. Fluctuations in circulating AVT levels do not correlate with pulses; and injections of AVT (at supraphysiological levels) elicit only minute urea-N pulses. However, circulating 5-hydroxytryptamine (5-HT) levels fluctuate considerably and physiological doses of 5-HT cause large urea-N pulse events. When the efferent cranial nerves to the gills are sectioned, natural urea pulse events persist, suggesting that direct motor output from the CNS to the gill is not the proximate control.

Adaptation, Physiological↗
PubMed2002

Diabetes worsens pulmonary diffusion in heart failure, and insulin counteracts this effect.

Chronic heart failure (CHF) (hydrostatic stress) and diabetes (basal laminae thickening) share the potentiality of damaging the alveolar-capillary membrane. We investigated 15 control subjects and 3 groups of 15 patients each having type 2 diabetes (Group 1), CHF (Group 2), and diabetes and CHF (Group 3), to probe whether addition of diabetes worsens lung diffusion in CHF and whether insulin counteracts this effect. Compared with control subjects, carbon monoxide diffusing capacity (DL(CO)) and diffusing capacity of the alveolar-capillary membrane at rest were increasingly depressed from Group 1 through Group 3. DL(CO) was lower than predicted in 11 patients each in Groups 1 and 2 and in all patients in Group 3. Regular insulin (10 IU) was ineffective in CHF alone, whereas it improved DL(CO) and diffusing capacity of the alveolar-capillary membrane in diabetes; changes, however, were significantly greater in the patients with both diabetes and CHF (+17.6%, +27.3%) than in those with diabetes alone (+9.2%, +13.1%). Insulin did not affect lung spirometry, volumes, and hemodynamics. Thus, gas transfer is depressed in a number of patients with diabetes or CHF; comorbidity increases the frequency and extent of this disorder. Insulin facilitates diffusion in diabetes, through an influence on alveolar-capillary conductance, and its efficacy is greater in comorbidity; diabetes is more disturbing in patients with CHF and produces a synergistic rather than a simple additive effect.

Aged↗
PubMed2006

The role of counter-current exchange in preventing hypoxia in skeletal muscle.

Mathematical models that describe oxygen transport from a single capillary into a region of surrounding tissue often predict that the tissue is hypoxic, whereas in reality diffusion from more richly perfused nearby capillaries prevents hypoxia from forming in the tissue. In this manuscript, a mathematical model of oxygen transport is presented that is applicable to vascular beds consisting of a large number of non-uniformly perfused parallel capillaries arranged in a manner characteristic of skeletal muscle. The model is used to examine conditions under which counter-current flow and myoglobin-facilitated diffusion provides sufficient oxygen to poorly perfused regions to prevent the occurrence of hypoxia. The method developed here leads to a coupled system of nonlinear ordinary differential equations for the oxygen concentration in the capillaries, and is easy to apply even for vascular beds containing a large number of capillaries.

Capillaries↗
PubMed1999

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.

Amidophosphoribosyltransferase↗
PubMed2004

A model for the mediation of processivity of DNA-targeting proteins by nonspecific binding: dependence on DNA length and presence of obstacles.

A physical and mathematical model is presented to explain processivity of proteins on DNA. In this model, a DNA-targeting protein such as a restriction enzyme can diffuse to the DNA surface and nonspecifically bind to it. Once on the DNA surface it will either move along the DNA or equilibrate with the surrounding region. Owing to the nonspecific binding, the search for a specific site on the DNA occurs in a reduced dimensionality, and the protein appears processive when moving from one specific site to another. The simplest version of this nonspecific-binding-facilitated diffusion model is solved and the results quantitatively explain experimentally observed dependence of the processivity ratio on the intervening DNA length between two specific sites.

Binding Sites↗