The Uptake Process

For analysis of glucose uptake in muscle it is convenient to divide the process into three steps. The first step is transfer of sugar from the capillary to the cell surface second, transport of sugar through the cell membrane and third, phosphorylation of glucose within the cell. Each step limits uptake to some extent under all conditions, but the degree of limitation imposed by an individual step is variable under various experimental conditions. 

The membrane theory of insulin action was strengthened by the work of l evine and associates (1949, 19.50 Levine and Goldstein, 1952) and Park and associates (1955, 1956) and has focused attention on the question of how glucose enters the c.e.11 in muscle tissue. 

The erythrocyte has proved to be a useful experimental object for permeability studies since it is relatively easy to separate glucose metabolism from membrane permeability and to measure the latter by both rapid optical methods and chemical analysis. From the work of Wilbrandt and colleagues (1947, 1956 Wilbrandt and Rosenberg, 1950 Wilbrandt, 1954), 

In this system, glucose is postulated to form a complex with a carrier molecule at the outer surface of the cell. The sugar-carrier complex passes across the membrane and releases free glucose at the inner surface. The process is reversible. The maximum transport rate, Tmax, is limited by fixed properties of the system such as the total number of carriers and their movement. Below this limit, however, transport will vary with the sugar concentration since this determines the extent of complex formation according to Langmuir adsorption or Michaelis-Menten kinetics. Thus unidirectional transport into the cell can be expressed as follows  

Studies of Membrane Transport Using Skeletal Muscle 

---

Carboxylic ionophores selectively transport cations by using intramolecular complexation in the uptake process of cations (basic region). A new ion transport system has been developed which incorporates a structural device which assists in the release process by using intramolecular complexation of an [18]crown-6 ring and a primary ammonium ion 48>. The experimental conditions are shown in Fig. 7. All these com-... 

Several attempts were made to monitor the uptake of Zn as a function of time. As a first attempt, the cells were exposed to 30 ppm Zn at room temperature. The rate of uptake of the Zn by the cells was much faster than was the case with Sn it was impossible to measure an increase in zinc content of the cells above that of the initial Zn -exposed sample either in florescence signal or GFAA signal by the methods described in this work. Reducing both the concentration of Zn in solution (10 ppm and 1 ppm) and the temperature (0°) did not slow down the uptake process enough to monitor an increase in accumulated Zn. By addition of known concentrations of Zn to the cells exposed to a 1.0 ppm Zn solution, it was found by GFAA that the amount of zinc accumulated... 

Feedback inhibition of amino acid transporters by amino acids synthesized by the cells might be responsible for the well known fact that blocking protein synthesis by cycloheximide in Saccharomyces cerevisiae inhibits the uptake of most amino acids [56]. Indeed, under these conditions, endogenous amino acids continue to accumulate. This situation, which precludes studying amino acid transport in yeast in the presence of inhibitors of protein synthesis, is very different from that observed in bacteria, where amino acid uptake is commonly measured in the presence of chloramphenicol in order to isolate the uptake process from further metabolism of accumulated substances. In yeast, when nitrogen starvation rather than cycloheximide is used to block protein synthesis, this leads to very high uptake activity. This fact supports the feedback inhibition interpretation of the observed cycloheximide effect. 

In the first case the mechanisms are based on an increased reducing capacity of Fe(lll)-chelates, a necessary step in the uptake process, with a concurrent increase in acidification and release of organic acids into the rhizosphere in the latter case molecules having high affinity for Fe (phytosiderophores) are synthesized and released into the rhizosphere when Fe is lacking. 

Figure 1.21, like Figure 1.19, shows two Schild plots, one of which (open circles) departs greatly from the expected behavior. The deviation occurs when noradrenaline is the agonist and again it can be accounted for in terms of the reduction in local concentration caused by the uptake, process... 

FIGURE 1.20 Hypothetical concentration-response curves to illustrate how the uptake) process can influence the study of the antagonism of noradrenaline by phentolamine. The two full lines show the response to noradrenaline, first in the absence and then in the presence of phentolamine. If the experiment is repeated, but with the uptake process blocked, the dotted lines would be obtained. Noradrenaline has become more active, and phentolamine now causes a greater shift (compare the lengths of the two horizontal arrows), as explained in the text. 

The logic of the evolution of insertion can now be considered. Much as the most primitive selectivity of the chemistry of the uptake process, pumping, carrying and final binding to form a useful enzyme is not an invention by organisms but is a necessary consequence of inevitable equilibrium considerations (see Section 4.17), so the binding of particular metal ions to particular chelatase proteins was similarly selected... 

The series of molecular events responsible for the uptake process constitutes the endocytic pathway, which enables cells to internalize macromolecules from the cell exterior, forming an endosome. The endosome is an intermediate organelle that serves as an essential component for many receptor-mediated signaling pathways and as a transport vector for eventual delivery to a specialized organelle known as the lysosome. Once in the lysosomal lumen, digestive enzymes provide essential metabolites from these macromolecules (i.e. free amino acids and lipids) directly to the cytosol for their use. 

The uptake process is energy-dependent since it can be inhibited by incubation at a low temperature or by metabolic inhibitors. The energy requirements reflect a coupling of the uptake process with the Na+ gradient across the... 

The free energy of the phosphorylated histidine (P His) or cysteine (P Cys) is comparable with the free energy of PEP (AG° = — 61.5 kJ mol ). The reactions (1) to (4) are therefore fully reversible under physiological conditions, whereas reaction (5) is irreversible. The substrate when bound to the domain IIC (or IID) obtains the phosphoryl group from the unit IIB, via unit IIA, which is rephosphorylated by P HPr. Efficient translocation of carbohydrates depends on the phosphorylated IIB domain. The release of the phosphorylated substrate terminates the uptake process. 

The main objective of this section is to examine the implications of the different transport mechanisms on the kinetics of the uptake process. Section... 

It is perhaps wise to begin by questioning the conceptual simplicity of the uptake process as described by equation (35) and the assumptions given in Section 6.1.2. As discussed above, the Michaelis constant, Km, is determined by steady-state methods and represents a complex function of many rate constants [114,186,281]. For example, in the presence of a diffusion boundary layer, the apparent Michaelis-Menten constant will be too large, due to the depletion of metal near the reactive surface [9,282,283], In this case, a modified flux equation, taking into account a diffusion boundary layer and a first-order carrier-mediated uptake can be taken into account by the Best equation [9] (see Chapter 4 for a discussion of the limitations) or by other similar derivations [282] ... 

Bioaccumulation is a complicated process that couples numerous complex and interacting factors. In order to directly relate the chemical speciation of an element to its bioavailability in natural waters, it will be necessary to first improve our mechanistic understanding of the uptake process from mass transport reactions in solution to element transfer across the biological membrane. In addition, the role(s) of complex lability and mobility, the presence of competing metal concentrations and the role(s) of natural organic ligands will need to be examined quantitatively and mechanistically. The preceding chapter... 

Although Theory and Modeling is more extensively discussed in Chapter 3, it is helpful to briefly discuss some basic concepts related to the accumulation of chemicals by SPMDs. Huckins et al. (1993) have shown that the uptake process obeys first-order kinetics (Figure 2.3). This type of exchange kinetics is characterized by half-lives (ti/2), which are constant for a particular set of conditions and... 

This stage of the uptake process is therefore called the equilibrium sampling phase . 

Changes of molecular conformations of the sorbate during the uptake process [23]. 

Apart from the diffusion step in the particle, when the uptake process occurs from a binary or multicomponent fluid mixture, there maybe an additional resistance to mass transfer associated with the transport of solutes through the fluid layer surrounding the particle. The driving force in this case is the concentration difference across the boundary layer, and the flux at the particle surface is... 

Figure 7.33 The renal accumulation and toxicity of gentamycin (G). Gentamycin is filtered in the glomerulus and enters the tubular lumen. Here, it is taken up by proximal tubular cells and in vesicles as part of the uptake process. These fuse with lysosomes (L) inside the cell. The accumulation of gentamycin inside the lysosome destabilizes it, causing it to rupture and release its hydrolytic enzymes (o). These cause damage within the cell. Also, gentamycin can directly damage mitochondria (M).

The calculated and experimental curves deviate considerably in shape, and it seems that the simple diffusion model is not adequate to describe the kinetics of the uptake process in this case. Also the value of D (9.25 X 10"9 cm.2/sec.) required to make the two curves coincide at early times seems to be much smaller than expected. The only data available on the diffusion of molybdenum oxide in liquid silicates is from a report by Norman et al. (11), who measured the diffusion constant of... 

The correspondence between the calculated and measured curves in the case of Te02 uptake by the clay loam was poor. Figure 4 shows that there is a tendency for the rate of uptake to be large at first and then to decrease abruptly, and in some cases reach a pseudo-equilibrium. This produces a rather pronounced bend in the experimental curves which is not present in the calculated curve. It appears that there was relatively rapid condensation of the Te02 onto available sites at or near the surface, and that after these have been saturated, the uptake process decreases markedly. 

The simple definition of biological availability as the fraction of the total trace metal available for uptake by the biota implicitly assumes that all relevant organisms will have similar uptake characteristics. In addition the use of biological availability as a chemical parameter involves the assumption that it can be identified with particular chemical species or groups of species. Perhaps surprisingly many of the studies carried out to date have lent support to these assumptions, though the detailed mechanisms of the uptake processes remain unclear. Some of the inorganic chemical species identified as... 

Before dyeing with oxidation dyes, the furs are treated with the appropriate killing agents and then mordanted with metal salts. Iron, chromium, and copper salts, alone or in combination, are used for mordanting, and the uptake process requires several hours. Adjustment of the pH is effected with formic, acetic, or tartaric acid. The final dyeing process is carried out in paddles with the precursors and hydrogen peroxide until the actual dye lake is developed and adsorbed within the hair fiber. It takes quite a few hours at room temperature until the dyeing process is finished. 

The uptake process was followed by purging for 7 min, and subsequently the TPD was carried out. The results were obtained by monitoring the decrease, during desorption, of the absorbance A measured in arbitrary units (au), of a typical IR band of the adsorbate molecules, where A is proportional to N (A = K x N), and where N is the amount of adsorbate in the material and K, a proportionality constant [98,99], For benzene, the region between 1450 and 1550 cm-1 was integrated to obtain a measure of the intensity of the band around 1482 cm-1. For toluene and ethylbenzene, the segment between 1477 and 1517 cm-1 was integrated to obtain an intensity measure for the band around 1497 cm-1 [97,98],... 

Energy-dependent, carrier-mediated uptake of thyroid hormone has been demonstrated in a variety of other cell types, including fibroblasts [109,110], pituitary tumor cells [111] and muscle cells [112]. In addition to the uptake processes in the plasma membrane, indirect evidence has been reported recently for active transport of T3 between subcellular compartments, i.e., from the cytoplasm to the nu-... 

Another process, tubular reabsorption, also takes place in the kidneys. Specific tubular uptake processes exist for carbohydrates, amino acids, vitamins etc. Drugs may pass from the tubule into the plasma if they are substrates for the uptake processes, or if they are lipid soluble (this process is highly dependent on the prevailing pH, see Section 1.3.4.2). 

---