Concanavalin A--Sepharose purification of soluble Na,K-ATPase from rectal glands of the spiny dogfish

Na,K-ATPase from the rectal glands of the spiny dogfish (Squalus acanthias) has been purified by concanavalin A—Sepharose affinity chromatography after solubilization in the nonionic detergent octaethyleneglycoldodecylmonoether. The method is rapid and yields enzyme at high protein concentrations, and the enzyme is fully active. The enzyme particles behave as a homogeneous population of particles, each containing protein, lipid, and detergent. The size of the particle is identical to what has been measured previously, giving a protein molecular weight of 270,000 with 50 mol of lipid bound.     

Retention of enzyme activity by detergent-solubilized sarcoplasmic Ca2+ -ATPase.

The Ca2+ -activated ATPase of sarcoplasmic reticulum can exist in true solution in the presence of some nonionic detergents, with retention of enzymatic activity for several days. The soluble active particles retain about 30 mol of phospholipid per mol of polypeptide chain even in the presence of a large excess of detergent, indicating the existence of relatively strong attractive forces between protein and lipid, as previous work from other laboratories has already suggested. Deoxycholate is much more effective than nonionic detergents in removing protein-bound lipid and, when used at solubilizing concentrations, completely delipidates and inactivates the ATPase. Preliminary molecular weight measurements indicate that the Ca2+ -ATPase exists as an oligomer in the native membrane: fully active enzyme in Tween 80 has a minimal protein molecular weight of about 400 000, corresponding to a trimer or tetramer of the ATPase polypeptide chain, and even the inactive enzyme in deoxycholate contains a substantial fraction of dimeric protein.     

Voluntary food consumption and gastric emptying in rats given intravenous Triton WR-1339

Intravenously administered Triton WR-1339, a nonionic surface active agent, has been used as an endogenous hyperlipemic agent since 1951. We expected Triton to increase food consumption to supply, at least partially, the energy and acetyl groups necessary for producing the hyperlipemic state. In this study, however, we observed that the rats injected intravenously with various dose levels of Triton decreased their voluntary food intake in a dose-related manner. Two other nonionic surface active agents, Tween 20 and Tween 80, given intravenously did not alter food intake. Further studies revealed that Triton WR-1339 administered intravenously 30 min before feeding by stomach tube resulted in a marked delay in the rate of gastric emptying which was also dose related. A delay in gastric emptying has previously been suggested as one mechanism that controls food intake. Tween 20 and Tween 80 did not alter the rate of gastric emptying. We suggest that the mechanism responsible for the decrease in voluntary food consumption in Triton WR-1339 injected rats may be due to the delay of gastric emptying in these animals.     

Influence of Formulation and Processing Variables on Properties of Itraconazole Nanoparticles Made by Advanced Evaporative Precipitation into Aqueous Solution

Nanoparticles, of the poorly water-soluble drug, itraconazole (ITZ), were produced by the Advanced Evaporative Precipitation into Aqueous Solution process (Advanced EPAS). This process combines emulsion templating and EPAS processing to provide improved control over the size distribution of precipitated particles. Specifically, oil-in-water emulsions containing the drug and suitable stabilizers are sprayed into a heated aqueous solution to induce precipitation of the drug in form of nanoparticles. The influence of processing parameters (temperature and volume of the heated aqueous solution; type of nozzle) and formulation aspects (stabilizer concentrations; total solid concentrations) on the size of suspended ITZ particles, as determined by laser diffraction, was investigated. Furthermore, freeze-dried ITZ nanoparticles were evaluated regarding their morphology, crystallinity, redispersibility, and dissolution behavior. Results indicate that a robust precipitation process was developed such that size distribution of dispersed nanoparticles was shown to be largely independent across the different processing and formulation parameters. Freeze-drying of colloidal dispersions resulted in micron-sized agglomerates composed of spherical, sub-300-nm particles characterized by reduced crystallinity and high ITZ potencies of up to 94% (w/w). The use of sucrose prevented particle agglomeration and resulted in powders that were readily reconstituted and reached high and sustained supersaturation levels upon dissolution in aqueous media.     

Anionic micelles and vesicles induce tau fibrillization in vitro

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of recombinant tau can be induced by treatment with various agents, including phosphotransferases, polyanionic compounds, and fatty acids. Here we characterize the structural features required for the fatty acid class of tau fibrillization inducer using recombinant full-length tau protein, arachidonic acid, and a series of straight chain anionic, cationic, and nonionic detergents. Induction of measurable tau fibrillization required an alkyl chain length of at least 12 carbons and a negative charge consisting of carboxylate, sulfonate, or sulfate moieties. All detergents and fatty acids were micellar at active concentrations, due to a profound, taudependent depression of their critical micelle concentrations. Anionic surfaces larger than detergent micelles, such as those supplied by phosphatidylserine vesicles, also induced tau fibrillization with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate tau fibrillization, that this mechanism underlies the activity of fatty acid inducers, and that anionic membranes may serve this function in vivo.     

Asymmetric distribution of anionic sites on rat liver nuclear membranes.

The labelling characteristics of isolated rat liver cell nuclei was studied using polycationized ferritin as an ultrastructural probe for anionic sites. At low concentrations of the marker the nuclear surface was partly labelled eg. at sites of nuclear annuli. At high probe concentrations the entire cytoplasmic surface of the outer nuclear membrane bound ferritin particles. On the other hand, the cisternal surfaces of nuclear membranes could not be labelled although in parallel experiments Concanavalin A-ferritin bound to the cisternal surface of both nuclear membranes indicating free access of ferritin particles to the perinuclear space. The results indicate that nuclear membranes show a distinct vectorial asymmetry in respect to the presence of anionic surface sites.     

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin was investigated by measuring the amount released of a marker dye from the vesicles. In solutions of pH around 7, anionic and nonionic surfactants caused vesicle disintegration at very low concentrations, while cationic surfactants produced a breakdown of the vesicles at rather high concentrations. Increase in the alkyl chain-length of surfactant molecules brought about decrease in the surfactant concentration at which vesicle disintegration starts. As the length of the polyoxyethylene chain in nonionic surfactant molecules increased, the tendency of vesicle disintegration to occur decreased. Both anionic and cationic surfactants gave clear solutions above their critical micelle concentrations when they acted on the phospholipid vesicles, whereas nonionic surfactants left ghost cell-like debris consisting of carboxymethylchitin molecules in their micellar solutions. The effect of pH on vesicle disintegration was notable for ionic surfactants but not for nonionic surfactants. Thus, anionic surfactants increased the degree of disintegration as pH increased, while cationic surfactants produced an identical vesicle disintegration curve below pH 8 above which the curve started to shift toward the lower concentration region of the agents. These findings were explained in terms of surfactant penetration into phospholipid bilayers and solubilization of phospholipid molecules by surfactant micelles.     

The nucleocapsid of the lipid-containing double-stranded RNA bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.

The nucleocapsid of the enveloped double-stranded RNA bacteriophage phi 6 was isolated by extraction with the nonionic detergent Triton X-114 and subjected to disruption analysis with chelating and protein-denaturing agents. The subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. The role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.     

Observations on Membranes of Mycoplasma laidlawii Strain B

The cytoplasmic membrane of Mycoplasma laidlawii strain B is solubilized by anionic and nonionic detergents, succinylation, phospholipase A, alkaline phosphatase, trypsin, and chymotrypsin. Cationic detergents are without effect, as are chelating agents, even in the presence of high concentrations of monovalent cation. The detergent-solubilized membrane exhibits one peak in the analytical ultracentrifuge, but the sedimentation coefficient is dependent upon concentration of detergent. Simple dialysis does not remove all of the sodium dodecylsulfate except from lipid-depleted membrane particles. Membranes bind sodium dodecylsulfate but acetone powders of membranes do not. Sulfated alcohols with chain lengths of C(14) and C(16) are more tightly bound than dodecylsulfate. A constant amount of di- and trivalent cation is bound by the membrane upon aggregation. Only a portion of this cation is removable with chelating agents. No chelating agent is bound by these aggregates. A portion of the lipid-depleted membrane particles is solubilized by negatively charged lipids and detergents, giving rise to aggregates in the presence of divalent cation. Fractionations of detergent-solubilized membranes by preparative gel electrophoresis and ammonium sulfate were inconclusive. Density gradient centrifugation of succinylated membranes yielded at least five fractions which exhibited homogeneity by ultracentrifugation. Analytical gel electrophoresis of these fractions demonstrated heterogeneity. The composition of these five fractions suggested separation of protein from lipid.     

Membrane Fusion and Rupture in Liposomes: Effect of Biodegradable pH-Sensitive Surfactants

Biodegradable pH-sensitive surfactants (BPS) are a unique family of easily metabolized compounds that demonstrate pH-dependent surface activity. These agents, in combination with other delivery systems, have demonstrated effects in enhancing transnucleic acid activity. The increased activity has been hypothesized to occur from a release of endosomal contents. Simply, the BPS delivery system containing nucleic acids enters the cell through an endocytotoic process. It encounters an acidic pH and becomes surface active leading to defects in the endosomal membrane. In the current study, an in vitro model membrane was used to better understand the liposome defect mechanisms that BPS elicit. Using this system, it is shown that BPS can induce both liposome fusion and rupture depending upon the pH and mole ratio of BPS to membrane lipids. Futhermore, liposome fusion induced by BPS was dependent on the total numbers of liposome particles while rupture was independent of interacting liposome particles. The generated data indicate that BPS agents act differently from other typical surface active agents and fuosgenic compounds. Instead of facilitating membrane fusion through the hexagonal II phase, BPS appeared to contribute and participate in the membrane fusion at different stages. Received: 18 February 1998/Revised: 14 July 1998     

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.     

Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Intracellular Ca2+ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini.

The association of 125I-labelled epidermal growth factor (125I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca2+ concentrations. These agents included cholecystokinin-octapeptide (CCK8) and the Ca2+ ionophore A23187. Inhibition by CCK8 was blocked by lowering the incubation temperature from 37 degrees C to 15 degrees C. Moreover, in contrast with studies of intact acini, the binding of 125I-EGF to isolated acinar membrane particles was not affected either by CCK8, or by varying the level of Ca2+ in the incubation medium. These results indicated, therefore, that the inhibition of 125I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37 degrees C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the 125I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca2+ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca2+-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca2+. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca2+ ionophore A23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca2+ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.     

Effect of detonation nanodiamonds on phagocyte activity

Detonation ND (nanodiamond) holds much promise for biological studies and medical applications. Properties like size of particles, inclination for modification of their surface and unambiguous biocompatibility are crucial. Of prime importance is interaction between ND and immune cells, which supervise foreign intrusion into an organism and eliminate it. Neutrophils are more reactive in inflammatory response implementing cytotoxical arsenal including ROS (reactive oxygen species). The aim of the work was to estimate the ability of two ND samples (produced by Diamond Center and PlasmaChem) to keep the vitality of neutrophils from the inflammatory site. The ability of cells to generate ROS in the presence of ND particles is considered as indicating their biocompatibility. IR spectra and size of particles in the samples were characterized. Acid modification of ND was carried out to get the luminescent form. In the biological aspect, ND demonstrated up or down action, depending on the concentration, time and conditions of activation of cells. Weak action of ND in whole blood was obtained possibly owing to the ND adsorbed plasma proteins, which mask active functional groups to interact with the cell membrane. ND did not influence the viability of isolated inflammatory neutrophils in low and moderate concentrations and suppressed it in high concentrations (≥1 g/l). Addition of ND to the cell suspension initiated concentration-dependent reaction to produce ROS similar to respiratory burst. ND up-regulated response to bacterial formylpeptide, but up- and down-modified (low or high concentrations, accordingly) response to such bacterial agents as OZ (opsonized zymosan), which neutrophils swallow up by oxygen-dependent phagocytosis. Localization of the particles on the cell surface as into the cells was identified by monitoring the intrinsic fluorescence of oxidized ND. The various mechanisms that could account for penetration of ND particles into the cell are discussed. Common conclusion concerns compatibility of ND with living neutrophils from inflammatory site and their normal functioning for infection safeguard.     

The action of tributyltin on energy coupling in coupling-factor-deficient submitochondrial particles

1. Tributyltin at concentrations of approx. 1nmol/mg of protein induces respiratory control and lessens the protein permeability of coupling-factor-deficient submitochondrial particles. 2. At these concentrations or lower, it increases the P/O ratio of the particles to a small extent and inhibits the adenosine triphosphatase activity without greatly increasing its sensitivity to uncoupling agents. 3. It fails to stimulate ATP-driven reversed electron transport or transhydrogenase, but stimulates the transhydrogenase driven by aerobic succinate oxidation. 4. The results indicate that, unlike oligomycin, tributyltin does not discriminate between damaged and intact ATP-synthesizing complexes. 5. The relationship between the oligomycin- and tributyltin-binding sites is discussed.     

Effect of surface-active agents on Na, K-ATPase from guinea pig kidneys

The effect of surface active agents on the activity of Na, K-ATPase and on the direct medium 18O exchange in the membrane preparations of the guinea pig kidney has been studied. The medium 18O exchange was considered as a character of the K+-dependent stage in ATPase reaction. Low concentration of all the surface active agents were shown to stimulate, and high concentrations -- to inhibit both ATPase and medium 18O exchange. The relation between medium 18O exchange and Na,K-ATPase activity was found to be equal to 1.5 +/- 0.1. The treatment of preparations by activating amounts of the surface active agents resulted in lowering this relation up to 1.0 +/- 0.09 and 1.3 +/- 0.04 for DOC and triton X-100, correspondently, due to a stronger stimulation of ATPase activity than of medium 18O exchange. With the inhibiting amounts of triton X-100 and histone H2a, this relation did not change, but it decreased in the presence of equal amounts of DOC.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents

Treatment of isolated human erythrocyte membranes with Triton X-100 at ionic strength ?0.04 preferentially released all the glycerolipid and glycoprotein species. At low ionic strength, certain nonglycosylated polypeptides were also selectively solubilized. The liberated polypeptides were free of lipids, but some behaved as if associated into specific oligomeric complexes. Each detergent-insoluble ghost residue appeared by electron microscopy to be a filamentous reticulum with adherent lipoid sheets and vesicles. The residues contained most of the membrane sphingolipids and the nonglycosylated proteins. The polypeptide elution profile obtained with nonionic detergents is therefore nearly reciprocal to that previously seen with a variety of agents which perturb proteins. These data afford further evidence that the externally-oriented glycoproteins penetrate the membrane core where they are anchored hydrophobically, whereas the nonglycosylated polypeptides are, in general, bound by polar associations at the inner membrane surface. The filamentous meshwork of inner surface polypeptides may constitute a discrete, self-associated continuum which provides rather than derives structural support from the membrance.     

Chelating Agent Shock of Bacteriophage T5

When two strains of phage T5 (heat-susceptible form T5st(+) and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed "chelating agent shock" (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st(+) was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.     

A detergent-activated tyrosinase from Xenopus laevis. I. Purification and partial characterization

A tyrosinase has been purified from the skin of the frog Xenopus laevis. Dihydroxyphenylalanine oxidase and tyrosine hydroxylase activities co-purify throughout the procedure. The enzyme is isolated in an inactive form, but both enzymatic activities are activated by a variety of anionic detergents. Of these, sodium dodecyl sulfate (NaDodSO4) is the most effective. The enzyme activation occurs at NaDodSO4 concentrations well below the critical micelle concentration and it remains active at concentrations as high as 30 mM (1%). Neither activity is stimulated by cationic or nonionic detergents, or a variety of other agents, including trypsin. The purified tyrosinase is a glycoprotein having a polypeptide Mr = 175,000 by NaDodSO4-polyacrylamide gel electrophoresis. This monomeric species is enzymatically active in the presence of NaDodSO4. Detergent-activated tyrosinase has a KM for dihydroxyphenylalanine of 6 X 10(-4) M and a KM for tyrosine of 4 X 10(-4) M. Both activities are inhibited by copper chelators but not by an iron chelator. Further characterization of the detergent activation of this enzyme is presented in a companion paper (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12542-12546).