Sulfate Transport System Research Articles

Anion transport across the red blood cell membrane mediated by dielectric pores.

The anion transport across the red blood cell membrane is assumed to occur by ionic diffusion through dielectric pores which are formed by protein molecules spanning the red blood cell membrane. The access of anions to the dielectric pores is regulated by anion adsorption sites positioned at the entrances of the pores. The adsorption of small inorganic anions to the adsorption sites is facilitated by ionizing cationic groups setting up a surface potential at the respective membrane surfaces. Applying the transition state theory of rate processes, flux equations for the unidirectional flux were derived expressing the unidirectional flux as a function of the fractional occupancies of anion adsorption sites at both membrane surfaces. The basic properties of the transport model were investigated. The concentration-dependence and the pH-dependence of the unidirectional fluxes were shown to depend upon the surface charge density and upon the affinity of the transported anion species to the anion binding sites. The concentration-response and the pH-response of the unidirectional fluxes of different anion species may differ substantially even if the anion species are transported by the same anion transport system. The model predicts a characteristic behavior of the Lineweaver-Burk plot and of the Dixon plot. A comparison between computer simulated and experimentally determined flux curves was made. By choosing a suitable set of parameters, the anion transport model is capable of simulating the concentration-dependencies and the pH-dependencies of the unidirectional sulfate and chloride flux. It is sufficient to change one single constant in order to convert the “sulfate transport system” into a “chloride transport system”. Furthermore, the model is capable of predicting the inhibitory action of chloride on the sulfate transport system. No attempts were made to fit the experimental data to the model. The behavior of the model was qualitatively in accordance with the experimental results.

The interaction of chloride with the sulfate transport system of Ehrlich ascites tumor cells.

The effect of Cl- on SO4(-2) efflux was studied in both Cl--containing and Cl--free ascites tumor cells loaded with 35SO4(-2) to test the hypothesis that Cl--SO4(-2) exchange is mediated by the same mechanism responsible for SO4(-2)-self exchange. The addition of Cl--free, 35SO4(-2) loaded cells to a SO4(-2)-free, Cl- medium results in: (1) SO4(-2) efflux that is dependent on the extracellular Cl- concentration (Km = 4.85 mM; ke = 0.048 min-1 at 50 mM Cl-) and (2) net Cl--uptake that exceeds SO4(-2) loss. Both SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and ANS (1-anilino-8-napthalene sulfonate) inhibit S04(-2) efflux but are without effect on Cl- uptake. The addition of Cl--containing, 35SO4(-2) loaded cells to a SO4(-2)-free, Cl- medium results in: (1) a slight gain in cellular Cl- and (2) ke for SO4(-2) efflux identical to that for Cl--free cells.

Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.

Transinhibition kinetics of the sulfate transport system ofPenicillium notatum: Analysis based on an iso Uni Uni velocity equation

Inorganic, intracellular sulfate, selenate, and molybdate strongly transinhibit the activity of the sulfate transport system in a mutant ofPenicillium notatum lacking ATP sulfurylase. All three Group VI anions are substrates of the sulfate transport system. No effect was observed on the activity of the choline-O-sulfate transport system. Choline-O-sulfate transport was transinhibited only by intracellular choline-O-sulfate. The results confirm the earlier conclusions of Bradfieldet al. (Plant Physiol.46:720, 1970) and Bellengeret al. (J. Bacteriol.96:1574, 1968) that transinhibition results from the action of the unchanged, internal substrate, and not a metabolite. The sulfate transport system is quite sensitive toL-cysteine.L-cysteine may be a feedback inhibitor of the sulfate transport system, or, alternately, some component of the sulfate transport system may be chemically (and nonspecifically) inactivated by reducing agents. Internal unlabeled sulfate, selenate, and molybdate acted as mixed-type inhibitors with respect to35SO42− transport, in agreement with the simple Iso Uni Uni model. The kinetic constants for sulfate are:Kms=1.25×10−5M,Vmax=2.5 μmoles×g−1×min−1,Kmp=2.5×10−3m,Kiip=5×10−3m. The apparent equilibrium ration of internal-to-external sulfate was about 104 at low initial substrate concentrations. External unlabeled 10−2m sulfate promotes exchange at a rate of 0.15 μmoles×g−1×min−1. DNP (10−4m) and anaerobiosis promote a small efflux. DNP inhibits the exchange reaction.

Uptake and efflux of sulfate in Neurospora crassa

Sulfate efflux from an intracellular pool was observed with both wild-type and cys-11 cells of Neurospora and apparently occurs by way of the sulfate transport system. Efflux requires the presence of external sulfate or the related ions, chromate, selenate, or thiosulfate, and probably occurs by an exchange reaction. The sulfur amino acids, cysteine or methionine, do not promote sulfate efflux. The K m for efflux is much greater than the K m for sulfate uptake, which permits the accumulation of a considerable intracellular pool before efflux becomes significant. Substantial transmembrane movement of sulfate both influx and exit, was found to occur in azidetreated cells, although the net uptake of sulfate was abolished by this inhibitor. Both sulfate uptake and efflux are inhibited by p- chloromercuribenzoate which suggests that the sulfate permease possesses an essential sulfhydryl group.

Regulation of sulfate transport in neurospora by transinhibition and by inositol depletion

Neurospora possesses two distinct sulfate transport systems, a low-affinity form (Permease I) which is the only type found in conidia, and a second species (Permease II) which predominates during the mycelial stage. Although methionine represses the synthesis of both of these permeases, inorganic sulfate only partially represses the mycelial form and does not affect the synthesis of Permease I. Both transport systems are also regulated by transinhibition. The transinhibition which occurs in mycelia is not due to an intracellular pool of inorganic sulfate, but is instead exerted by an early intermediate of the sulfate assimilatory pathway. The development of functional sulfate transport activity depends upon genetic and metabolic events which affect the cell membrane. The synthesis of sulfate permease activity in the inos mutant requires an exogenous supply of inositol. The effect of the cot mutant, which is thought to interfere with membrane synthesis, also prevents the development of sulfate permease at the restrictive temperature. The maintenance of pre-existing functional sulfate permease activity apparently also requires a continuous renewal of membrane components since withdrawal of inositol from inos mutants results in a rapid inactivation of transport activity.

Evidence for a sulfate transport system in Escherichia coli K-12

Evidence for a sulfate transport system in Escherichia coli K-12

The specific interaction of chromate with the dual sulfate permease systems of Neurospora crassa

Chromate inhibits growth and eventually causes cell death in Neurospora crassa. Mutants resistant to chromate show stage-specific deficiencies in sulfate transport and parallel losses in chromate uptake. The toxicity of chromate is completely dependent upon the sulfur source provided for growth. The mutant strains are chromate-resistant when methionine is present but are very sensitive to the analog when inorganic sulfate serves as the sole sulfur source. Chromate is a competitive inhibitor of sulfate transport and the K i for inhibition (3 × 10 −5 m) is nearly identical to the K m for chromate entry. The transport of chromate is energy dependent, inhibited by sulfate, repressed when growth occurs on methionine-supplemented media, and clearly occurs via the sulfate permease systems. The toxicity of chromate seems to include two aspects: (1) specific inhibition of sulfate transport and perhaps early assimilatory steps, and (2) general lethality due to entry and strong oxidizing activity of chromate. The chromate-resistant mutants largely exclude the analog and thus are protected from the generalized toxic effect. Tungstate and molybdate show a similar, but far weaker, inhibition of the sulfate transport systems and at higher concentrations cause a parallel inhibition of growth in all strains.

Location of sulfate-binding protein in Salmonella typhimurium.

A method is described for location of proteins in bacteria. It depends upon two techniques. One technique is the inactivation of the protein by a reagent which is incapable of penetrating the bacterial membrane (permeability barrier). Proteins inside this membrane cannot be inactivated unless the cells are disrupted; proteins on or outside the membrane can be inactivated. The second technique depends upon inactivation of the protein by specific antibody. Antibody should not penetrate the external bacterial wall, and therefore should only inactivate proteins that are on the wall surface. Thus, proteins can be localized inside the membrane, in the wall-membrane area, or outside the wall. One reagent developed for use with the first technique is diazo-7-amino-1,3-naphthalene-disulfonate. It inactivated beta-galactoside transport, but not beta-galactosidase of intact Escherichia coli. Similarly, it inactivated sulfate binding and transport but not uridine phosphorylase activity of Salmonella typhimurium. This indicates that the sulfate-binding protein is on or outside the cell membrane, and that uridine phosphorylase is inside the cell. The organic mercurial compounds used also showed that the sensitive parts of the sulfate and alpha-methylglucoside transport systems are less reactive than the sensitive part of the beta-galactoside system. Antibody to the sulfate-binding protein inactivated the purified protein but did not inactivate this protein when intact bacteria were employed. Thus, it appears that the sulfate-binding protein does not protrude outside the cell wall. The conclusion that the binding protein is located in the wall-membrane region is supported by its release upon spheroplast formation or osmotic shock, and also by its ability to combine with sulfate in bacteria which cannot transport sulfate into the cell.

Biochemical Studies on Active Transport

The active transport process, so important in cell function, has been studied in the past with intact cells. Models which have arisen from this work all depend on: first, a specific protein to recognize the substrate; second, translocation of the substrate across the cell membrane; third, release of substrate within the cell and restoration of the system to its initial state. These steps are adequate for facilitated transport, but in active transport an energy input is required to maintain a concentration gradient. Parts of transport systems have been isolated recently. A protein which specifically recognizes ß-galactosides has been partially purified. In another case, a protein that appears to be the recognition part of the sulfate transport system of Salmonella typhimurium has been crystallized, and many of its properties have been described. The role of this protein in recognition and in translocation is discussed. Also proteins that phosphorylate a variety of sugars as they enter the cell's interior provide a mechanism for concentrating sugars as their phosphates, against a gradient.

The inorganic sulfate transport system of Penicillium chrysogenum

Inorganic sulfate enters the mycelium of Penicillium chrysogenum against an apparent concentration gradient by a temperature-, energy-, pH-, and concentration-dependent transport system that is regulated by the degree of sulfur-deficiency of the mycelium. Sulfate transport can take place in the absence of sulfate reduction and organic sulfur formation. The rapid conversion of sulfate to organic sulfur in the absence of N-ethylmaleimide suggests that the newly-transported 35SO 4 −− does not completely equilibrate with the pre-existing sulfate pool. Only about half of the transported sulfate could be removed by exhaustive washing or exchanged with unlabeled extracellular sulfate. The kinetics of transport and exchange also suggest the presence of two distinct intracellular sulfate pools. Inorganic thiosulfate, a suggested intermediate and overflow product of fungal sulfate reduction is an extracellular inhibitor of sulfate transport. Sulfite is an extracellular inhibitor at pH 5 but not at pH 6.5. Methionine and cysteine are not extracellular inhibitors of sulfate transport, but are rapidly converted intracellularly to a feedback inhibitor. The V max and apparent K m of the sulfate transport system (in the presence of NEM) are 0.5 – 1.0 μ mole/gmminute and 3–7 × 10 −5 m, respectively, at pH 6.5. Inorganic selenate inhibits sulfate transport and is taken up by the mycelium via the sulfate transport system. In the absence of net transport, sulfate will still adsorb to the mycelial surface very rapidly (or penetrate into a free space) by a DNP- and temperature-insensitive process.