William H. Habig's research while affiliated with National Institutes of Health and other places
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Publications (71)
The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases α. β, γ, δ and e an the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dod...
Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline...
The potency tests for bacterial vaccines are quite diverse. For some products (pertussis, cholera, anthrax, typhoid and BCG vaccines) these are specified as Additional Standards in the Code of Federal Regulations. For other products (tetanus and diphtheria toxoids, plague vaccine) the testing is done according to so-called Minimum Requirements, whi...
A single dose of 0.25 ng of tetanus toxin (TeTx), equivalent to approximately 5 minimal lethal doses, injected intracerebrally to 1-day-old rats, caused translocation, i.e., activation, of Ca(2+)-phosphatidylserine-dependent protein kinase C (PKC) from the cytosolic to the membrane compartment within 1 h. Six hours after treatment with the toxin, a...
Diphtheria toxin (DT) can translocate across endosomal membranes in response to low pH. Buried hydrophobic domains localized in the 37-kDa toxin B chain become exposed in response to acidic conditions and are thought to participate in the membrane translocation process. The crystal structure of DT has revealed a structurally distinct translocation...
Botulinum toxin is a protein neurotoxin produced by Clostridium botulinum. Botulinum toxin blocks neurotransmission at the neuromuscular junction by inhibiting the presynaptic release of acetylcholine (for a recent review, see ref. 1). The ability of botulinum toxin to block neurotransmission and paralyze or weaken muscles has been useful in the tr...
Administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP vaccine) or endotoxin (LPS) resulted in marked alterations in hepatic drug-metabolizing enzymes in endotoxin-responsive (R) and non-endotoxin-responsive (NR) mice. A single human dose (0.5 ml) of DTP vaccine increased hexobarbital-induced sleep times to 1.6- to 1.8...
Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD5...
We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug...
Administration of Diphtheria and Tetanus Toxoids and Pertussis Vaccine Adsorbed (DTP) vaccine to mice causes dose- and time-dependent alterations in hepatic drug metabolism as determined by hexobarbital-induced sleep time and several direct measurements of soluble and microsomal enzyme activities. Vaccines containing only tetanus and/or diphtheria...
The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 μg/ml, tetanus toxin blocked stimulated rel...
A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the...
The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was ma...
Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaf...
Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacte...
The extent of immunological similarity between tetanus toxin and botulinum toxins A, B, C1, and E was studied by using 10 antibodies produced against synthetic peptides representing different sequences of tetanus toxin, mouse antitetanus serum, and human Tetanus Immune Globulin. Antibodies produced against the synthetic peptides recognized tetanus...
• Slipped capital femoral (SCFE) occurs with greater frequency in children with growth hormone deficiency than in children in the general Population. This epidemiologic observation suggests that SCFE may be a complication of growth hormone deficiency, associated pituitary hormone deficiencies, growth hormone therapy, or a combination of these facto...
The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin...
Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialoganglios...
We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex. Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-a...
Tetanus toxoid elicits protective antibodies against tetanus toxin in humans and animals. It has been reported that antitoxin from immunized humans contains no anti-light chain antibodies, based on immunodiffusion and quantitative precipitin analyses. We confirmed the absence of precipitating anti-light chain antibodies in tetanus immune globulin....
We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material a...
The effects of phenytoin (30 micrograms/ml), phenobarbital (64 micrograms/ml), and carbamazepine (24 micrograms/ml) were assessed in cerebral cortical cell cultures. After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid a...
2-Propylthiouracil has been reported as replacing glutathione as a substrate for the glutathione transferases of rat liver. This observation has been examined with several homogeneous glutathione transferases that were prepared from human and rat liver by different methods in three laboratories. No evidence was obtained for 2-propylthiouracil as a...
A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin. This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain. These results show that the structural gene for the toxin is on the plasmid. The pCL1...
125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB...
Tetanolysin produced similar rates of leakage of K+ and hemoglobin from erythrocytes. When studied by using cholesterol-containing black lipid membranes, this hemolysin induced conductance steps with a broad frequency distribution. These findings are inconsistent with the formation of structural channels and suggest that tetanolysin acts by causing...
Monoclonal antibodies specific for fragment B, fragment C, and light chain of tetanus toxin were prepared by fusion of P3X63Ag8 BALB/c myeloma cells with spleen cells from BALB/c mice immunized with tetanus toxoid or fragment B. Hybridoma colonies were assayed for antibody production by an enzyme-linked immunosorbent assay. Fourteen positive clones...
The effects of direct application of tetanus toxin on fetal mouse spinal cord neurons in culture are described. Tetanus toxin produces increased excitation characterized by paroxysmal depolarizing events (PDE). In contrast to the abrupt onset of convulsant action produced by postsynaptic glycine antagonist strychnine, the convulsant action of tetan...
Blockade of spontaneous electrical activity in dissociated fetal spinal cord cultures produced neuronal deficits as measured by biochemical and morphological techniques. Spinal cord cultures exhibited an age-dependent vulnerability to impulse blockade with tetrodotoxin (TTX) or xylocaine. Neuronal cell counts, [125I]tetanus toxin fixation and [125I...
Tetanolysin binding to lipid vesicles was found to depend on the molar ratio of cholesterol to phospholipid, being low in vesicles containing up to 20 mol% cholesterol and high in vesicles containing more than 33 mol%. High concentrations of purified tetanolysin preparations formed arc- and ring-shaped structures. The structures were not readily de...
The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When a...
The binding characteristics of 125I-labeled tetanus toxin and 125I-labeled fragment C to rat brain membranes have been studied. Fragment C is a proteolytically derived portion of the holotoxin. Tetanus toxin is slightly more potent than fragment C in displacing 125I-labeled tetanus toxin bound to rat brain membranes. Conversely, fragment C is more...
Rat cerebral neurons maintained in monolayer culture accumulate 125I-labeled tetanus toxin. Accumulation is receptor-mediated; i.e. it can be prevented by including unlabeled tetanus toxin, gangliosides, or tetanus antitoxin in the incubation medium but not by including tetanus toxoid, high concentrations of serum, or thyrotropin. Accumulation is t...
Publisher Summary This chapter presents a procedure for the preparation of glutathione transferases of the rat and the human. The glutathione S-transferases are the enzymes catalyzing conjugation reactions with glutathione as the first step in mercapturic acid synthesis. Although these enzymes may be distinguished from each other by their character...
Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite r...
Hara et al. [3] reported 'that after treatment of cultures of C. [Clostridium] tetani with various agents, nontoxigenic derivatives could be isolated with a far higher frequency than could be expected for the occurrence due to mutations in bacterial chromosomes.' They suggested that the genetic factor controlling the production of tetanus toxin mig...
Fragment C of tetanus toxin appears to retain most of the determinants of the tetanus toxin molecule insofar as interactions with neural or thyroid membranes and with gangliosides are concerned. Thus, unlabeled fragment C is nearly as effective as unlabeled tetanus toxin in inhibiting the binding of either 125I-labeled toxin or 125I-labeled thyrotr...
Tetanus toxin isolated from Clostridium tetani and radioiodinated by both the Hunter-Greenwood and the Bolton-Hunter procedures was analyzed by isoelectric focusing on Polyacrylamide gel and by “quantitative” gel electrophoresis. Immediately after iodination, the focused preparation exhibited a single protein component with a pI′ of 5.1, while afte...
Accumulation of the permeant lipophilic cation [³H]tetraphenylphosphonium (TPP⁺) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K⁺ and by veratridine. A further 1:10 to 1:15 decrease in TPP⁺ uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomyci...
Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent...
125I-labeled tetanus toxin interacts with the glycoprotein component of the thyroid thyrotropin receptor when this component is in solution or when it is incorporated into a liposome. Binding can be inhibited by both unlabeled thyrotropin and tetanus toxin but not by unlabeled prolactin, glucagon, insulin, ACTH, or growth hormone; binding can also...
Glutathione transferase ϱ has been purified to homogeneity from human erythrocytes. The enzyme has a molecular weight of 47,500 and is composed of two subunits of the same apparent molecular weight. The enzyme is active in catalyzing the reaction of glutathione, as a nucleophile, with a variety of compounds bearing an electrophilic center. Thioethe...
Normal rat thyroid membranes adsorb neurotoxicity when incubated with purified tetanus toxin. Membranes from a rat thyroid tumor with a thyrotropin receptor defect adsorb very little neurotoxicity when similarly evaluated. This inability of the tumor membranes to adsorb neurotoxicity is correlated with a defect in their ability to bind both 125I-la...
The catalyzed reactions of GSH with organic nitrate and thiocyanate esters and with a series of chloronitrobenzene substrates have been investigated and the results used to formulate a mechanism for glutathione S-transferase catalysis. All the homogeneous preparations of the glutathione transferases that have been tested catalyze the reaction of GS...
Glutathione S-transferase AA from rat liver was purified to apparent homogeneity as judged by gel filtration and gel electrofocusing. The protein has an isoelectric point near pH 9.9 and a molecular weight of 45,000 and is composed of two apparently identical subunits. The enzyme is most active with 1-chloro-2,4-dinitrobenzene and glutathione as su...
The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with...
Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intri...
The conjugation of glutathione to prostaglandin A1 is susceptible to enzymatic catalysis.
WE report direct evidence that conjugation with glutathione (GSH) is a significant mechanism for detoxification of the epoxides of polycyclic aromatic hydrocarbons. We believe that the level of the enzymes may be directly relevant to the carcinogenic potential of the hydrocarbons, which are common pollutants1. They are metabolised by microsomal mix...
Evidence is presented that glutathione S-transferases, a group of enzymes active in the formation of thioethers from glutathione and a large number of compounds with an electrophilic carbon atom, can also catalyze the formation of nitrous acid and oxidized glutathione from organic nitrate esters such as nitroglycerin. In addition, organic thiocyana...
Glutathione transferase A was purified from rat liver. The enzyme catalyzes the conjugation of glutathione with compounds bearing an electrophilic site, especially those in which the electrophilic site is on, or α to, an aromatic ring. The enzyme has a molecular weight of 45,000 and is composed of 2 similar subunits. Initial velocity, product inhib...
The purification of homogeneous glutathione S transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in whi...
Glutathione transferase A has been purified from rat liver. The enzyme catalyzes the conjugation of glutathione with compounds
bearing an electrophilic site, especially those in which the electrophilic site is on, or α to, an aromatic ring. The enzyme
has a molecular weight of 45,000 and is composed of two similar subunits.
Initial velocity, produ...
Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodo...
The purification of homogeneous glutathione S-transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in whi...
Phycocyanin, a chromoprotein from Chroomonas sp., is characterized in regard to its size, subunit structure, amino acid composition, and spectroscopic properties. It
is a monodisperse protein of 50,000 daltons and is composed of two polypeptide chains of 10,000 and two chains of 16,000 daltons.
The proposed structure of the "native" protein is α2β2...
Glutathione transferases catalyze the conjugation of GSH with a number of compounds bearing a nucleophilic group. The number and specificity of such enzymes from rat liver were surveyed. Three enzymes have been purified to homogeneity; each was active with p-nitrobenzyl chloride as substrate. One was also active with epoxides and p-nitrophenethyl b...
Citations
... The activity of chitinase was assayed according to (Bade & Stinson, 1981). The Glutathione S-transferase (GST) activity was determined according to (Habig et al., 1974). ...
... The results are expressed in U CAT/mg protein, where one unit of CAT was defined as the amount of enzyme that catalysed the dismutation of 1 μmol of H 2 O 2 /min. Glutathione S-transferase (GST) activity was measured spectrophotometrically using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) as described by Habig et al. (1974). GST activity is expressed as nmol/min/mg protein. ...
... The particularity of group III C. botulinum strains is that the neurotoxin genes, bontC and bontD, are localized on phages which are not integrated into the chromosome [22][23][24][25]. Thus bont-harboring phage can be easily lost during C. botulinum cultures which become no longer toxic. ...
... GR activity was measured as NADPH oxidation in the presence of 0.2 mM NADPH as described by Murshed et al. (2008b). The conjugation of GSH with excess 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm (ε340 = 0.0096 μM −1 cm −1 ) was used to measure GST activity (Habig et al. 1974). All measurements of absorbance were made with ELISA Microplate reader (Synergy Mx, BioTek Instruments Inc., USA). ...
... For glutathione-S-transferase (GST) activity, leaf tissue was extracted in potassium phosphate buffer (0.2 M; pH 7.5). The activity of GST was determined by the methodology of Habig and Jakoby (1981) using substrate 1-chloro-2,4-dinitrobenzene (Handa et al. 2019). Protein content was estimated as per Bradford (1976) method as detailed in Rai and Agrawal (2021). ...
... Les glutathion-S-transférases (EC : 2.5.1.18) ont un rôle important dans la détoxication des substances xénobiotiques exogènes ou endogènes (Sau et al., 2010), en catalysant la conjugaison de ces substances avec le groupement thiol du glutathion endogène (Jakoby & Habig, 1980;Chelvanayagam et al., 2001;Boyer, 2006;Walters et al., 2009;Ebadollahi et al., 2013). Le rôle majeur des GSTs est F o r P e r s o n a l U s e 82 de convertir des composés lipophiles en molécules hydrophiles facilement excrétables (Habig et al., 1974). ...
... Given the inclusion of cytosol containing glutathione S-transferases in these studies, it was deemed necessary to evaluate the catalytic activity of these cytosolic transferases toward glutathione conjugation to a "model" substrate. A review of the literature revealed that 1-chloro-2,4-dinitrobenzene serves as a substrate, to varying degrees, for all 6 of the glutathione transferases isolated from rat liver (Jakoby et al., 1976). ...
... Antioxidants superoxide dismutase (SOD) by Mishra and Fridovich (1972) and Concetti (1976) Catalase (CAT) by Beers and Sizer (1952). Red cell reduced glutathione (GSH) by Habig (1974) glutathione peroxidase (G-Px) by Paglia (1967) Hexokinase by King (1965). Na + -K + ATPase by Ismail and Edelman (1985) Glycosylated hemoglobin by Eross (1984) Hexose by Niebes (1972). ...
... Unlike intact animal systems, long term studies are readily performed (Habig et al., 1986); the toxin is not cytotoxic. While the phenomena of toxin action have been well characterized (Bergey et al., 1981, 1983, 1987; H-abig et al., 1986), to better characterize the effects of various conditions and agents on toxin action and recovery better means for quantification of the action of the toy'1 are needed. Later investigations reported here focused on the application of a system for continuous detection of postsynaptic potentials to this experimental system to provide quantification of synaptic effects of the toxin. ...
![[object Object]](https://i1.rgstatic.net/ii/profile.image/442126371692545-1482422549612_Q64/Gregory-Bergey.jpg)
... The phycocyanin is produced in a limited number of microalgal species, while Spirulina sp. is generally considered a quite suitable algal species. Other species, such as Chroomonas sp., Synechococcus sp., Anabaena oryzae, and Nostoc muscorum, are also potential candidates for phycocyanin production (Abd et al., 2018;MacColl et al., 1973). Phycocyanin synthesis is affected by several inducible factors, e.g., light conditions, nitrogen restriction, and metabolic substrate concentration (Boussiba and Richmond, 1980;Manirafasha et al., 2018). ...
