Use of a genetic variant to study the hexose transport properties of human skin fibroblasts.

Human skin fibroblasts from 'normal' subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the 'normal' and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.     

Antigen-specific stimulation of amino acid transport in bovine lymphocytes

Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD-B) from this organism induced an increase in the transport of α-aminoisobutyric acid (AIB) and α-methylaminoisobutyric acid (MeAIB). PPD-B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two-fold enhancement after 20 hours, and continued to increase to 30- to 40-fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na⁺-dependent, A-system, and leucine transport was mostly by Na⁺-independent system(s). In contrast, AIB transport was about 25% by the A-system, 25% by at least one Na⁺-dependent, non-A-system, and 50% by one or more Na⁺-independent system(s). Analysis of the three components of AIB transport after treatment with PPD-B showed that: (1) transport by both the A-system and the Na⁺-independent system(s) was stimulated; (2) A-system transport was stimulated to a larger extent than Na⁺-independent transport; and (3) Na⁺-dependent, non-A-system transport was not stimulated significantly.     

2-Deoxy-D-glucose resistant yeast with altered sugar transport activity

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2-deoxy-D-glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the Vmax values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.     

Inhibition of active strontium transport from erythrocyte ghosts by internal calcium: Evidence for a specificity controlling site

Summary The inhibition of strontium transport from erythrocyte ghosts by internal calcium was investigated. When active strontium transport was measured in the presence of increasing levels of internal calcium it was found that the inhibition of strontium transport started at an internal calcium level of 0.3mm and was virtually complete when this concentration reached 1.0mm. It was also noted that calcium transport was virtually constant between concentrations of 0.3 and 1.0mm. This experiment indicated that calcium did not inhibit strontium transport by competing for the active site of the transport system. This inhibition was partially reversed by increasing the internal magnesium concentration from 1 to 4mm. A higher level of magnesium at the time of lysis and during incubation enhanced strontium transport. However, the inhibition remained noncompetitive with respect to calcium. Manganese was also found to support calcium and strontium transport. However, it could not reverse the inhibition of strontium transport by internal calcium at any concentration tested. In fact, manganese restored the inhibition of strontium transport by calcium in ghosts that were prepared and incubated in solutions that had high magnesium levels.     

Methionine transport in Pseudomonas aeruginosa.

A high-affinity (Km = 2.7 x 10(-7) M) energy-requiring methionine-transport system has been characterized in RM 46 and RM 48, two different PAO methionine auxotrophs of Pseudomonas aeruginosa. After 8 s of transport 40--60% of the methionine label in the alcohol extract appears in S-adenosyl-L-methionine (SAM) with the remaining activity in free methionine. Methionine transport required a high degree of structural specificity for transport. Stimulation of transport occurred by addition of glucose or organic acids. The ability of a given substrate to stimulate transport was related to the type of carbon source used for growth. Transport was sensitive to sulfhydryl reagents and required oxidative phosphorylation, as indicated by the inhibitory effects of anaerobiosis, cyanide, and arsenate. The degree of inhibition by arsenate correlated with the level of ATP in the cell. Rapid transport in a SAM-deficient mutant (TM 1) and inhibition by arsenate of transport in this mutant suggested that SAM formation was not directly linked to transport and that ATP supplied energy for transport. Inhibition by arsenate was more severe in glucose- compared to citrate-stimulated cells. This result was also observed with proline transport indicating that this was not a peculiarity of the methionine-transport system. These data emphasize the close link between glucose metabolism, ATP levels, and transport. This ATP level is not so critical for transport in cells metabolizing citrate.     

Transport of glucose, gluconate, and methyl alpha-D-glucoside by Pseudomonas aeruginosa

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-d-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K(m) of 7 muM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl alpha-d-glucose (alpha-MeGlc) via the glucose transport system. The apparent K(m) of alpha-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for alpha-MeGlc than for glucose. While only unchanged alpha-MeGlc was detected intracellularly in P. aeruginosa, alpha-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.     

Hexose transport after glucose refeeding of glucose-starved human fibroblasts: 1. The effects of tunicamycin and cycloheximide. 2. Insulin binding and action

Hexose transport in glucose-starved human fibroblasts was readily reversed by glucose refeeding. This hexose transport reversal was not inhibited by tunicamycin (1.5 microgram/ml) but was blocked by cycloheximide (20 micrograms/ml). The ability of insulin (100 mU/ml) to stimulate hexose transport was returned by glucose refeeding and this was not affected by tunicamycin. Cycloheximide which blocked the glucose refeeding effect on hexose transport, decreased the ability of insulin to stimulate hexose transport. Specific 125I-insulin binding was increased by glucose refeeding of glucose-starved cells and this change in binding was inhibited by tunicamycin and cycloheximide. Thus, it appears that under the conditions employed in human fibroblasts, the ability of insulin to stimulate hexose transport is differentially regulated more by factors affecting basal hexose transport than by those affecting changes in insulin binding.     

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.     

l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems

The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K_m for high affinity transport was 30 micromolar. The K_m for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p-chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers.     

Inhibition by amiloride of sodium transport into rabbit kidney medulla microsomes

Sodium transport into rabbit kidney medulla microsomes was 50% inhibited by amiloride. This Na⁺ uptake was shown to represent transport when the uptake process was reversed by the ionophore nigericin. The transport was complete within 60 min and proportional to the microsomal protein concentration. The effect of amiloride on transport was specific since the similar compound sulfaguanidine failed to affect microsomal Na⁺ transport. Amiloride-sensitive Na⁺ transport into microsomes was inhibited 70% by decreasing the pH (from 7.0 to 5.9), but was unaffected by the presence of a pH gradient. The kinetics of Na⁺ transport could be explained by a simple model, assuming that amiloride lowered the rate of Na⁺ entrance into the vesicles but had no effect on the rate of efflux. The failure of amiloride to effect efflux from the vesicles was also demonstrated directly.     

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.     

Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p-chloromer-curibenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D-glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system.     

Energy coupling of the hexose phosphate transport system in Escherichia coli

The active transport of hexose phosphates in Escherichia coli was inhibited by many uncouplers or inhibitors of oxidative metabolism. Fluoride and the lipid soluble cation, triphenylmethylphosphonium, had little effect. The uninduced level of transport was sensitive to fluoride, but not to azide. After energy uncoupling of active transport, the cells could equilibrate their intracellular water with the glucose-6-phosphate in the medium and displayed exit counter-flow suggesting the existence of carrier-mediated transport in the energy-uncoupled cells. The uncoupled transport of glucose-6-phosphate was inhibited by fructose-6-phosphate; the uninduced level of glucose-6-phosphate transport was not inhibited by fructose-6-phosphate. After energy uncoupling, the influx had a low affinity suggesting that, unlike the transport of beta-galactosides, the energy coupling for the active transport of hexose phosphate involved a change in the affinity of influx.     

Relationship between amino acid transport and electron transport by membrane vesicles of Micrococcus denitrificans

Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.     

A second function of the S gene of bacteriophage lambda

Infection of Escherichia coli by bacteriophage lambda caused an immediate inhibition of uptake by members of all three classes of E. coli active transport systems and made the inner membrane permeable to sucrose and glycine; however, infection stimulated alpha-methyl glucoside uptake. Phage infection caused a dramatic drop in the ATP pool of the cell, but the membrane did not become permeable to nucleotides. Infection by only one phage per cell was sufficient to cause transport inhibition. However, adsorption of phage to the lambda receptor did not cause transport inhibition; DNA injection was required. The inhibition of transport caused by lambda phage infection was transient, and by 20 min after infection, transport had returned to its initial level. The recovery of transport activity appeared to require a lambda structural protein with a molecular weight of 5,500. This protein was present in wild-type phage and at a reduced level in S7 mutant phage but was missing in S2 and S4 mutant phage. Cells infected with S7 phage had a partial recovery of active transport, whereas cells infected with S2 or S4 phage did not recover active transport. Neither the inhibition of transport caused by phage infection nor its recovery were affected by the protein synthesis inhibitors chloramphenicol and rifampin.     

Nickel transport by the thermophilic acetogen Acetogenium kivui

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.     

Nickel transport by the thermophilic acetogen Acetogenium kivui.

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.     

Damaging action of photodynamic treatment in combination with hyperthermia on transmembrane transport in murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin derivative caused inhibition of the 2-aminoisobutyric acid transport system. This was reflected by an increase in the apparent Km with a constant Vmax, indicating impairment of the carrier function rather than a decrease of the number of transport sites. Hyperthermic treatment of these cells resulted in a moderate decrease of the activity of the 2-aminoisobutyric acid transport system. Overall protein synthesis was severely inhibited both by photodynamic treatment and by hyperthermia. Hyperthermia subsequent to photodynamic treatment resulted in an additive inhibition of 2-aminoisobutyric acid transport and of protein synthesis. After photodynamic treatment both 2-aminoisobutyric acid transport and protein synthesis were repaired. The repair of 2-aminoisobutyric acid transport depended on protein synthesis, as shown by the virtually complete blockage of repair by anisomycin. After hyperthermia (either alone or subsequent to photodynamic treatment), no recovery of 2-aminoisobutyric acid transport was observed, although protein synthesis was restored to the initial level. Apparently, hyperthermia subsequent to photodynamic treatment blocks the repair of photodynamically induced damage of this transport system. The experimental results further indicate that protein synthesis is not the rate-determining step for the repair of 2-aminoisobutyric acid transport, although it is necessary in this process. Cell survival was decreased both by photodynamic treatment and by hyperthermia. The combined effects of these two treatments were additive. It is discussed that these results indicate that photodynamic inhibition of 2-aminoisobutyric acid transport is not causally related to loss of clonogenicity, contrary to earlier suggestions.     

Tryptophan Transport in Neurospora crassa II. Metabolic Control

The rate of tryptophan transport in Neurospora is regulated by the intracellular pool of tryptophan. When cells were shifted from growth in minimal medium to tryptophan-containing medium for 10 min, there was a 50% reduction in the rate of tryptophan transport. Intracellular tryptophan pools derived from indole were equally effective in reducing the rate of transport as externally supplied tryptophan. The regulatory influence of tryptophan on the transport system appears to be a property of all the amino acids transported by the tryptophan transport site or sites. Lysine and glutamic acid are not transported by the tryptophan transport site or sites and are ineffective in the regulation of tryptophan uptake. Continued protein synthesis is required for the maintenance of a functional tryptophan transport system. The half-life of the transport system, estimated by inhibiting protein synthesis with cycloheximide, was about 15 min. Turnover of the system occurred at 30 C but not at 4 C, suggesting that the breakdown of the system is enzymatically mediated. It was inferred that the rate of tryptophan transport in Neurospora is modulated through the maintenance of a delicate balance between the synthesis and breakdown of some component of the transport system.     

cGMP transport by vesicles from human and mouse erythrocytes

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.