Substrate Concentration Gradient Research Articles

Modelling Shoot:Root Relations: the Only Way Forward?

The basic transport-resistance (TR) model of shoot:root carbon:nitrogen allocation is described. This approach assumes that the two processes of substrate transport and chemical conversion determine allocation. It is suggested thatallallocation models, whether built for the purposes of theoretical investigation or practical application, should start with this irreducible framework. Here it is assumed that the processes operate according to: (a) for substrate sources, dependence on shoot and root sizes with possible product inhibition; (b) for transport, movement down a substrate concentration gradient; and (c) for substrate sinks or utilization, linear bisubstrate kinetics. Some dynamic properties of the model are explored. Failure of this approach to allocation flags the need for additional mechanisms to control the processes. Details of the failure will indicate the modifications needed, which may involve hormones or reflect teleonomy (apparently goal-seeking behaviour), and which are added to the irreducible framework. However, these additions should not replace the irreducible framework of transport and chemical conversions, because they do not in reality. Modifications to the basic model to represent possibilities such as ontogenesis with the transition from exponential growth towards a steady state or with the scaling of within-plant transport resistances with plant size, the influence of hormones, and active transport, are described.

Measurement of polyamine transport. Adherent cells.

Most cells can take up polyamines, and some polyamine analogs, by a process that is independent of amino acid transport systems (,), and that, in certain circumstances, can substitute for synthesis de novo. In some cells polyamines are taken up by both saturable and nonsaturable systems. Uptake by saturable systems is energy-requiring, temperature-dependent, carrier-mediated, and operates against a substantial concentration gradient (,). In many cells putrescine and, often, spermidine transport is sodium-sensitive (the term sodium-sensitive is preferable to the more frequently used sodium-dependent, because removal of extracellular sodium reduces, but often does not abolish, uptake). In contrast, spermine transport in most cells is not affected by changes in extracellular sodium concentrations. The number of carriers in the polyamine transport system varies with cell type. In human umbilical vein endothelial cells there are apparently two carriers, one shared by spermine and spermidine, and one capable of transporting all three polyamines (). In contrast, porcine aortic endothelial cells appear to possess three carriers, one for each polyamine (). Multiple carriers are not uncommon, although many cell types appear to possess a single polyamine carrier able to transport putrescine, spermidine, and spermine, although with differing affinities ().

Activated-sludge system design for species selection: analysis of a detailed multispecies model

We analyze a detailed activated sludge model to determine the boundaries for proliferation of floc formers during aerobic or anoxic selection and with soluble or solid substrates. The analysis of competition with several filamentous species shows that the outcome is similar to that of competition with a single species. For the two-species competition we present a general procedure for determining the boundaries of desired operation and compute these boundaries for a slightly modified version of the AEROFIL model (Kappeler and Gujer, 1994a, b Water Resources 28, 303–322). To achieve high conversions in aerobic reactors a substrate concentration gradient should be established implying that the recycle ratio and the sludge-age should be sufficiently small while the dissolved oxygen concentration should be sufficiently large. The analysis of two mixed aerobic reactors suggests that the selector volume should be comparable or larger than that of the reactor. Analysis of an anoxic selector coupled with an aerobic reactor showed that good selection can be obtained with small selectors and a sufficiently large recycle ratio. A control mode based on switching between these two regimes has been suggested. Sustaining the growth of floc formers on a solid-substrate feed that undergoes hydrolysis is a difficult task if the kinetic parameters of the hydrolyzed and soluble substrate are similar. This subject requires further experimental and modeling efforts to account for better selection that has been observed with solid substrates.

Microdialysis techniques in the study of brain and skeletal muscle.

Traditionally, plasma measurements have been used to monitor metabolic events and the actions of hormones that are actually taking place within tissue beds that are anatomically separated from the vascular compartment. It is generally assumed that the extracellular fluid (ECF) within metabolically active tissues is composed of substrates and hormones in concentrations that closely approximate those in plasma. Indeed, this view is implicit in non-steady-state tracer calculations. However, through the use of microdialysis techniques in the study of tissue metabolism this view is being challenged. Our data suggest that there may be substantial concentration gradients for a variety of fuels between plasma and ECF, i.e. fuels (e.g. glucose) removed from the circulation being lower and fuels (e.g. glycerol, lactate, some amino acids) produced by tissues being higher than plasma levels. In short, the metabolic milieu seen by individual tissues (and hormone receptors?) may, at least in some instances, be strikingly different from that in plasma, and as a result, plasma measurements by themselves may not appropriately define the contributions of specific tissues to metabolic events, and overlook the importance of metabolic processes which are largely restricted to individual tissue beds. Through the use of microdialysis as a means of directly sampling ECF from metabolically important body tissues and with the evolution of its use in animal and human research, this technique will continue to offer exciting new insights into tissue metabolism and to investigate fundamental issues that cannot be addressed by other methods.

Cytoplasmic transport of lipids: Role of binding proteins

After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back from the cell before they could reach their targets. Conversely, intracellular lipids generated by hydrolysis of triglycerides, phospholipids and other esters must be transported away from their site of formation to prevent toxic accumulation. Intracellular movement of all molecules is slowed by molecular crowding, tortuosity, and the greater viscosity of the cytosol relative to water. However, lipids and other amphipathic molecules are further slowed by their tendency to bind to cytoplasmic membranes. Cytoplasmic binding proteins reduce membrane binding by increasing the aqueous solubility of their ligands. These aqueous carriers catalyze the transport of lipid molecules across hydrophilic water layers just as plasma membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the principal features of carrier-mediated transport, including saturation, mutual competition, and countertransport. Higher concentrations of cytoplasmic binding proteins are associated with more rapid cytoplasmic transport of longchain fatty acids. Available data suggest that substantial intracellular concentration gradients of fatty acids should exist, and that these gradients may help determine which metabolic pathway the fatty acid enters. Thus, cytoplasmic carrier proteins may help regulate the uptake and metabolism of fatty acids and other lipid molecules.

An analysis of transport resistances in the operation of BIAcore™; implications for kinetic studies of biospecific interactions

The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion-reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore™ instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damköhler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occured in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.

The effects of exposure time and pressure on the temperature-programmed desorption spectra of systems with bulk states

Abstract A mean-field model describing temperature programmed desorption (TPD) spectra for coupled reaction-diffusion (RD) systems is introduced. This finite medium model includes two states; a surface state and a state in the bulk which supports concentration gradients. A unified model which treats both exposure and desorption during a typical TPD experiment is presented. The substantial concentration gradients developed in the bulk during exposure depend sensitively on the rate of exposure, and profoundly influence the spatiotemporal evolution of concentration profiles during TPD. Our approach follows the development of the experiment accurately and can be used to probe the detailed characteristics of coupled RD systems. The nominal exposure in Langmuir units (L) is not adequate for coupled RD systems, since diffusion into the bulk during adsorption depends strongly on time. The detailed exposure schedule used, including exposure pressure and exposure time, must be reported for the correct interpretation of the TPD spectra of RD systems.

Physical Model Relating Diffusional Transport and Concurrent Metabolism of Peptides in Metabolically Active Cell Sheets

□ The physical model developed describes the complex interplay of diffusion and saturable metabolism in metabolically active cell sheets under reflection kinetics in contact with a bulk solution containing a substrate. A prominent implication of this work is the enzymatic cleavage of drugs in the viable epidermis, which represents the major metabolic barrier of the skin. The mathematical solution is based on steady-state Fickian diffusion and nonlinear Michaelis-Menten kinetics. Both substrate concentration gradients in the cell sheets and metabolic rates versus substrate concentration profiles were generated by mathematical simulation. The effects of various parameter estimates on the overall kinetics were studied (i.e., cell sheet thickness, effective diffusion coefficient, cell sheet/bulk solution partition coefficient, and maximum metabolic rate). By variation of the parameters, shifts between metabolism control and diffusion control are predicted. As an example, metabolism-controlled substrate turnover in thin cell sheets transforms into diffusion control in thick cell sheets, as diffusion of fresh substrate into cell sheets of increasing thickness becomes rate limiting. Close approximation of experimental data concerning Ala-4-methoxy-2-naphthylamide and Leu-enkephalin metabolism in cell culture sheets was achieved by independent simulations. The model may thus help to evaluate the potential of metabolically labile drugs (e.g., peptides) to penetrate the metabolic barrier of the viable epidermis.

Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport

Whether amphetamine acts principally at the plasma membrane or at synaptic vesicles is controversial. We find that d-amphetamine injection into the Planorbis giant dopamine neuron causes robust dopamine release, demonstrating that specific amphetamine uptake is not required. Arguing for action at vesicles, whole-cell capillary electrophoresis of single Planorbis dopamine neurons shows that amphetamine reduces vesicular dopamine, while amphetamine reduces quantal dopamine release from PC12 cells by > 50% per vesicle. Intracellular injection of dopamine into the Planorbis dopamine neuron produces rapid nomifensine-sensitive release, showing that an increased substrate concentration gradient is sufficient to induce release. These experiments indicate that amphetamine acts at the vesicular level where it redistributes dopamine to the cytosol, promoting reverse transport, and dopamine release.

Functional studies of P-glycoprotein in inside-out plasma membrane vesicles derived from murine erythroleukemia cells overexpressing MDR 3. Properties and kinetics of the interaction of vinblastine with P-glycoprotein and evidence for its active mediated transport.

Active [3H]vinblastine (VBL) transport (efflux) was documented for inside-out plasma membrane vesicles from murine erythroleukemia cells (MEL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P-glycoprotein (P-gp) 80-fold. Uptake of [3H]VBL at 37 degrees C by these inside-out vesicles, but not rightside-out vesicles or inside-out vesicles from wild-type cells, was obtained in the form of a rapid, initial phase (0-1 min) and a slower, later phase (> 1 min). The rapidity of each phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR-specific phase was temperature- and pH-dependent (optimum at pH 7), osmotically insensitive, and did not require ATP. The second MDR-specific phase was temperature-dependent, osmotically sensitive, and strictly dependent upon the presence of ATP (Km = 0.37 +/- 0.04 mM). Although other triphosphate nucleotides were partially effective in replacing ATP, the nonhydrolyzable analogue ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time course appears to represent tandem binding of [3H]VBL by P-gp and its mediated transport, with the latter process representing the rate-limiting step. In support of this conclusion, both binding and transport were inhibited by verapamil, quinidine, and reserpine, all known to be inhibitors of photoaffinity labeling of P-gp, but only transport was inhibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [3H]VBL was 7-7.5-fold lower than the rate of binding (Vmax = 104 +/- 15 pmol/min/mg protein, Kon = 1.5 - 2 x 10(5) mol-1 s-1) to P-gp, each phase exhibited saturation kinetics and values for apparent Km and KD for each process were approximately the same (215 +/- 35 and 195 +/- 30 nM). Intravesicular accumulation of [3H]VBL was almost completely eliminated by high concentrations of nonradioactive VBL, suggesting that simple diffusion does not contribute appreciably to total accumulation of [3H]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-out vesicles under the conditions employed did not maintain a P-gp mediated pH gradient. However, ATP-dependent, intravesicular accumulation of osmotically sensitive [3H]VBL occurred against a substantial permeant concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process.

Functional expression of P-glycoproteins in secretory vesicles.

We expressed P-glycoproteins (P-gps) encoded by the three mouse mdr genes in the membranes of secretory vesicles (SV) accumulating in the yeast mutant strain sec 6-4. Expression of the Mdr1 and Mdr3 isoforms in SV membranes caused a significant increased accumulation of the drug vinblastine (VBL) over background levels measured in control SV. The Mdr1/Mdr3-mediated increased drug accumulation could be completely abolished by the P-gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulation over background levels. Mdr3-mediated VBL transport was not affected by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous proton-translocating PMA1 H(+)-ATPase responsible for the strong electrochemical membrane potential across SV membranes. Moreover, in the presence of a delta micro-H+ across the SV membranes (inside positive) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accumulation of the lipophilic cation and P-gp substrate tetraphenylphosphonium, suggesting that P-gp-mediated uptake of this cation occurs against an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a steep proton gradient (inside acid) and was independent of any proton movements, excluding a proton synport or antiport mechanism for P-gp-mediated drug transport. Finally, we could demonstrate that colchicine accumulation in Mdr3-expressing SV occurred against a significant substrate concentration gradient, reaching a 7-fold increase in intravesicular colchicine concentration above the extravesicular medium drug concentration. Our studies show that SV isolated from the temperature-sensitive yeast sec 6-4 mutants are an ideal tool to express and to functionally characterize heterologous membrane proteins, in general and P-gps, in particular.

Heterogeneous Distribution of Microbial Activity in Methanogenic Aggregates: pH and Glucose Microprofiles

Methanogenic aggregates, harvested from an upflow anaerobic sludge blanket reactor treating potato starch wastewater, were acclimatized to either glucose or a mixture of sugars and organic nitrogen compounds (i.e., diluted molasses). Both types of granules exhibited internal pH and substrate concentration gradients in mineral medium (pH 7.0, 30 degrees C) as was measured with microelectrodes. Glucose-acclimatized granules suspended in a mineral medium lacking glucose exhibited a distinct internal pH decrease of about 1 U within the granule, suggesting strong metabolism by the acidogenic bacteria. Molasses-acclimatized and aged granules suspended in mineral medium did not exhibit such a pH decrease, suggesting the importance of the metabolic state of these acidogens. The pH gradient did not occur in deactivated granules and was not observable in strongly buffered media (mineral medium containing 33 mM phosphate or reactor liquid). When glucose (0.5 to 5.0 mM) was added to the mineral medium, granules exhibited a convex pH profile. Glucose consumption was located exclusively in the outer 200 to 300 mum of the aggregates (mean diameter = 1.5 mm). The addition of 20 mM 2-bromoethanesulfonic acid to the mineral medium indicated that the higher pH levels in the centre of the granule appeared to be related to the activity of methanogens. It is suggested that acidogenic activity occurs predominantly in the outer 200 to 300 mum of the aggregate and methanogenic activity occurs predominantly in the center of the investigated granules.

Glucose, pyruvate, and lactate concentrations in the blastocoel cavity of rat and mouse embryos

The concentrations of glucose, pyruvate, and lactate have been measured in the blastocoel fluid of single rat and mouse blastocysts, using the technique of micropuncture combined with an ultramicrofluorescence assay. When cultured in the presence of 5.55 mM glucose, 11.5-12.5 mM L-lactate and 0.25-0.33 mM pyruvate, concentrations in the blastocoel fluid of mouse and rat were 2.30 and 2.75 mM glucose, 14.6 and 19.6 mM L-lactate, and 0.13 and 0.50 mM pyruvate, respectively. When cultured in the presence of 1.0 mM glucose and 1.0 mM L-lactate, concentrations in the blastocoel fluid were 0.50 and 0.59 mM glucose and 2.22 and 3.70 mM L-lactate, respectively. These results suggest that (1) the blastocyst is capable of maintaining considerable concentration gradients of substrates across the trophectoderm, (2) the microenvironment of the blastocoel is adequately supplied with energy substrates for the development of the inner cell mass, and (3) the inner cell mass is capable of developing in both high and low glucose and lactate concentrations.

A scale-down two-compartment reactor with controlled substrate oscillations: Metabolic response of Saccharomyces cerevisiae

Substrate concentration gradients are likely to appear during large scale fermentations. To study effects of such gradients on microorganisms, an aerated scale-down reactor system was constructed. It consists of a plug flow reactor (PFR) and a stirred tank reactor (STR), between which the medium is circulated. The PFR, which is an aerated static mixer reactor, was characterized with respect to plug flow behaviour and oxygen transfer. A Bodenstein number of 15–220, depending on residence time and aeration rate, and a kLa of 500–1130 h−1, depending mainly on aeration rate, were obtained. The biological test system used, was aerobic ethanol production by Saccharomyces cerevisiae, due to sugar excess. The ethanol concentration profile and the yield of biomass were compared in two fed-batch fermentations. In the first case, the feeding point of molasses was located at the inlet of the PFR. This simulates location of the feeding point in the segregated part of a heterogeneous reactor, with local high sugar concentrations. In the second mode of operation, as a control with good mixing conditions, the PFR was disconnected from the STR, into which the substrate was fed. Differences were found: Up to 6% less biomass was produced and a larger amount of ethanol was formed in the two-compartment reactor system, due to the uneven sugar concentration distribution. This emphasizes the importance of the location of, and the mixing conditions at, the feeding point in a bioreactor.

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.

Meiofauna and solute transport in marine muds

Meiofauna typically inhabit sedimentary zones having substantial concentration gradients in biogcochemically important solutes such as O2. Diffusion experiments with the conservative tracers Cl− and Br− demonstrate that natural populations of meiofauna (∼30–100 cm 3) can increase transport rates of these solutes by factors of ∼1.7–2.3 × (for T > 10°C) compared with uninhabited sediments. The effect varies seasonally, directly with temperature and probably with faunal composition. About 20–40% of the increase in transport is due to increased porosity (decreased tortuosity) caused by meiofaunal activities and would be accounted for in most estimates of diffusion. The remaining, larger portion, is apparently due more directly to biologically induced fluid motion and three-dimensional diffusion. Transport coefficients can be resolved into two components with apparent activation energies of ∼3 and ∼10 kcal mol−1 (Q10 ∼ 1.2 and 1.8, T = 10°–20°C), consistent with control by physical and metabolic processes. Measurements following addition of particular meiofaunal groups to otherwise natural populations suggest that nematodes, juvenile bivalves, and polychaetes have the greatest effect. Transport activities of meiofauna must stimulate solute fluxes and reaction rates, particularly aerobic decomposition and associated processes such as nitrification in the oxic zones of marine sediments. In contrast, macrofauna commonly enhance solute transport to a greater extent (2–10 ×) than do meiofauna and, while stimulating aerobic reactions, also dramatically promote anaerobic processes in organic-rich deposits.

Functional specialization of different hepatocyte populations.

Hepatocytes from the periportal (afferent) and perivenous (efferent) zones of the liver parenchyma differ in their enzyme content and subcellular structures and thus have different metabolic capacities. Therefore the model of metabolic zonation proposes a functional specialization for the two zones: 1) oxidative energy metabolism with beta-oxidation, amino acid catabolism, ureagenesis, gluconeogenesis for the synthesis of both glucose and glycogen, cholesterol synthesis, bile formation, and protective metabolism are predominantly located in the periportal zone; 2) glycolysis, glycogen synthesis from glucose, liponeogenesis, ketogenesis, glutamine formation, and xenobiotic metabolism are preferentially situated in the perivenous zone. The input of humoral and nervous signals into the two zones is different. During passage of blood through the liver acinus, concentration gradients of oxygen, substrates, and hormones are established; the nerve densities in the two zones seem to be different. The different expression of the genome in upstream and downstream hepatocytes can be caused among other factors by the zonal gradients in oxygen and hormone concentrations. The functional specialization of the different hepatocyte populations is especially well documented for carbohydrate, amino acid, ammonia, and xenobiotic metabolism as well as for bile formation by a number of different approaches. Zonal flux differences were calculated from enzyme and metabolite distributions measured in vivo. They were observed in periportal- and perivenous-like hepatocytes in cell culture and in hepatocyte populations enriched in periportal and perivenous cells. They were detected also during ortho-and retrograde liver perfusion and finally by noninvasive techniques, using surface micro-light guides and miniature oxygen electrodes. The peculiar zonal hepatotoxicity of many xenobiotics can be explained at least in part by the enzymatic and fine-structural hepatocellular heterogeneity.

Metabolic zonation of liver parenchyma.

Periportal and perivenous hepatocytes differ in their content of enzymes and subcellular structures and thus in their metabolic capacities. Therefore, the model of metabolic zonation proposes that: (1) periportal hepatocytes catalyze predominantly oxidative energy metabolism with beta-oxidation and amino acid catabolism as well as ureagenesis for glycogen synthesis and glucose release, bile formation with cholesterol synthesis and protective metabolism; and (2) perivenous hepatocytes mediate preferentially glucose uptake for glycogen synthesis, glycolysis, and liponeogenesis as well as ketogenesis, glutamine formation, and xenobiotic metabolism. Periportal and perivenous hepatocytes are under the control of a different input of humoral and nervous signals, because concentration gradients of oxygen, substrates, and hormones are established during passage of blood through the liver and because gradients of nerve densities seem to exist. In periportal and perivenous hepatocytes gene expression can be different due to the zonal gradients in oxygen and hormone concentrations as well as nerve densities. The functional specialization of periportal and perivenous hepatocytes has been demonstrated especially well for carbohydrate, amino acid and ammonia, and xenobiotic metabolism as well as for bile formation by different techniques: Calculation of metabolic rates based on the zonal distributions of enzymes and metabolites, measurements of rates in periportal-like and perivenous-like hepatocytes in cell culture and in hepatocyte populations enriched in periportal and perivenous cells as well as in perfused livers during orthograde and retrograde flow using standard methods and noninvasive techniques with surface microlight guides and miniature oxygen electrodes.

Site-directed mutagenesis and ion-gradient driven active transport: on the path of the proton.

The lac permease of Escherichia coli is a hydrophobic transmembrane pro­ tein, encoded by the lac Y gene, that catalyzes the coupled translocation of (3-galactosides with H+ (i.e. H+ -substrate symport or cotransport) (see 12, 13, and 31 for recent reviews). Thus when a H+ electrochemical gradient (AjLw) is generated across the cytoplasmic membrane (interior becoming negative and/or alkaline), the permease utilizes free energy released from the downhill translocation of H+ in response to AjLw to drive uphill accumula­ tion of (3-galactosides against a concentration gradient (Figure 1A). Con­ versely, when a concentration gradient of substrate is created in the absence of AjLw, the permease utilizes free energy released from the downhill trans­ location of substrate to drive H+ uphill with generation of AjLw. The polar­ ity of this electrochemical gradient depends on the direction of the substrate concentration gradient: When [substrate]in AjLw is interior positive and acid (Figure 18); when [substrate]in > [substrate]out. AjLJI+ is interior negative and alkaline (Figure Ie)]. The lac permease is a model sys­ tem for a wide range of biological machines that transduce free energy

Determinants of mitochondrial O2 dependence in kidney.

The O2 dependence of mitochondrial cytochromes was studied in suspensions of isolated rat kidney cells to examine the determinants of renal mitochondrial function. Direct spectroscopy of the oxidation of cytochromes c + c1, a + a3, and b561 + b566 showed that oxidation-reduction changes of the entire electron transport chain occur over the same range of O2 concentrations; this suggests that the mitochondrial cytochromes function as a single unit in response to O2 changes. Half-maximal oxidation (P50 value) of cytochrome c + c1 in intact cells was 3.6 microM but was only 0.45 microM in isolated renal cortex mitochondria under state 3 conditions. This is consistent with the existence of a substantial O2 concentration gradient from the extracellular space to the region around the mitochondria under hypoxic conditions. Comparison with results for digitonin-treated cells under state 3 conditions indicates that the distribution of mitochondria within the cellular structure is an important determinant of the cellular O2 dependence. In addition, the mitochondrial O2 dependence varies as a function of cellular respiration rate. Stimulation of the O2 consumption with either a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a sodium ionophore, nystatin, increased the P50 value. Conversely, inhibition of ATP consumption by Na+-K+-ATPase with ouabain, or inhibition of mitochondrial electron transport with antimycin A, decreased the P50 value. Thus the O2 concentration required for mitochondrial function in the kidney is affected by mitochondrial distribution within the cell as well as by the functional demands imposed on the cell.