William H. Habig’s research while affiliated with National Institutes of Health and other places

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Publications (72)


Multiple Forms of Human Glutathione S‐Transferase and Their Affinity for Bilirubin
  • Article

June 2008

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127 Reads

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17 Citations

European Journal of Biochemistry

Kazuaki KAMISAKA

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William H. HABIG

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Jeanne N. KETLEY

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[...]

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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 dodecylsulfate-gel electrophoresis. Transferases α, β and ɛ each appear as a single band an gel electrofocusing: transferases α and δ are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48 500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate


Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin

October 2006

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14 Reads

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36 Citations

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 buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Trissaline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GDlb≅ GTlb≅ GQ1b > GD2 > GD3≫ GD1a≅ GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.


Potency testing of bacterial vaccines for human use

December 1993

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28 Reads

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18 Citations

Veterinary Microbiology

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, which have less regulatory authority than Additional Standards. Still other products (e.g., polysaccharide conjugate vaccines, acellular pertussis vaccine, live oral typhoid) are tested according to individualized criteria that are contained in their specific Product License Applications. For some products there is inadequate knowledge of the pathogenic mechanisms and/or protective factors to design valid in vitro potency tests. In these cases, animal testing with subsequent serologic evaluation or challenge testing is often necessary. Examples would include vaccines such as cholera and plague vaccines. The FDA supports the elimination of animal testing when suitable alternatives are available. Thus, many of the potency tests, especially for newer products, rely on in vitro characterization. For example, the immunogenicity of conventional polysaccharide vaccines is largely proportional to their molecular weight. Potency testing therefore relies heavily on physical characterization in terms of composition, molecular weight, and quantity.


The role of proline 345 in diphtheria toxin translocation
  • Article
  • Full-text available

March 1993

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37 Reads

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37 Citations

Journal of Biological Chemistry

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 domain composed of nine alpha-helices with their interconnecting loops (Choe, S., Bennett, M., Fujii, G., Curmi, P., Kantardjieff, K., Collier, R., and Eisenberg, D. (1992) Nature 357, 216-222). Two of these alpha-helices, TH8 and TH9, are unusually apolar and constitute the central core of the translocation domain. It has been proposed that these domains and the highly charged interconnecting loop undergo a conformation change under acidic conditions producing a dagger-like structure capable of inserting into the membrane thus initiating the translocation process. Proline 345 occupies a strategic location at the end of the TH8 alpha-helix. Proline residues have the ability to undergo a cis-trans isomerization reaction and because of this have been proposed to play a role in the conformational change that is a prerequisite for toxin translocation. The role of the proline at position 345 in membrane translocation was investigated. Pro was mutagenized to Glu and to Gly using a two-step recombinant polymerase chain reaction procedure, and the mutant proteins were expressed in vitro. Glu, an alpha-helix former, and Gly, an alpha-helix breaker, were selected for mutagenesis to distinguish between a structural role for Pro as an alpha-helix breaker and alternative roles, perhaps involving cis-trans isomerization-related conformational changes. Replacing Pro at position 345 with Glu or Gly resulted in a 99% reduction in toxicity to Vero cells. The enzymatic and binding activity of the toxin were not altered by the mutations. Instead, the reduction in toxicity is due to decreased translocation ability, suggesting that the Pro at position 345 plays a specific role in toxin membrane translocation.

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GT1b Ganglioside Prevents Tetanus Toxin-Induced Protein Kinase C Activation and Down-Regulation in the Neonatal Brain In Vivo

March 1993

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29 Reads

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19 Citations

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 40-50% reduction in the total brain PKC (cytosolic plus membrane) activity was noticed. GT1b (2 micrograms per brain) ganglioside, a putative receptor for TeTx, completely prevented enzyme translocation when injected intracerebrally 30 min before toxin administration and abolished down-regulation after 6 h from the time of toxin injection. GM1 (2 micrograms per brain), a ganglioside of lesser affinity for TeTx, produced by itself a 20-30% reduction of the total PKC activity and did not reverse TeTx-induced PKC down-regulation after 6 h. 12-O-Tetradecanoylphorbol 13-acetate (TPA) phorbol ester, administered at a concentration of 5 x 10(-5) M, caused activation and down-regulation of the enzyme, although with several orders of magnitude lesser potency. GT1b prevented the TPA-induced down-regulation.


An Overview of Some Issues in the Licensing of Botulinum Toxins

January 1993

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4 Reads

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 treatment of different neurologic disorders. A number of focal dystonias and other conditions with involuntary muscle movements currently are being treated with botulinum toxin, and it appears to be a potentially valuable therapy for many of these conditions.2 A preparation of botulinum toxin type A (trade name Oculinum, manufactured by Allergan) was approved for human use by the Food and Drug Administration (FDA) in 1989. Oculinum is approved for the treatment of blepharospasm and strabismus, and also has been used experimentally for a number of other disorders. Oculinum has also been designated as an orphan drug under the Orphan Drug Act (ODA) of 1983, which entitles the manufacturer to a seven year period of exclusive marketing for the specific indication for which the product has been approved. This exclusive marketing granted to the manufacturer of Oculinum raises questions of how additional botulinum products can be licensed, for either the same approved indications as Oculinum or for other indications. This article will review general requirements for approval of botulinum toxin by the FDA, and discuss how regulations in the Orphan Drug Act might affect approval of additional botulinum toxin products.


Role of endotoxin in alterations of hepatic drug metabolism by diptheria and tenanus toxoids and pertussis vaccine adsorbed

October 1992

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16 Reads

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19 Citations

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-fold above those of controls in both strains of mice. This effect persisted for 7 days. In contrast, Bordetella pertussis LPS-treated mice showed an increase at 1 day (3.0-fold for R mice and 1.5-fold for NR mice), which returned to control levels by day 7. Furthermore, cytochrome P-450 levels were decreased 30 to 40% 24 h after DTP vaccine administration in both R and NR mice, while after LPS administration they were decreased 30% in R mice and less than 10% in NR mice. Both spleen and liver weights of R and NR mice were increased 7 to 14 days following DTP vaccine administration. However, LPS treatment had no apparent effect on liver weights, and spleen weights of R mice were elevated from days 3 to 7. Histopathologic tissue examination showed random, multifocal inflammation with hepatocyte necrosis after DTP vaccine administration to both R and NR mice and an absence of lesions in LPS-treated mice. Premixing LPS with polymyxin eliminated the increased sleep times, but premixing DTP vaccine with polymyxin did not affect the increased sleep times. Levels of tumor necrosis factor and interleukin-6 in plasma of R mice were markedly increased after DTP and LPS treatment, while NR mice had reduced increases. These results suggest that LPS contributes to the alterations in R and NR mice seen within the first 24 h of vaccine administration but that it is not likely to contribute to the effects observed at later time points.


Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice

February 1992

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6 Reads

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6 Citations

Vaccine

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 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.


The Effects of Interleukin 2 and α-Interferon Administration on Hepatic Drug Metabolism in Mice

February 1992

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12 Reads

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32 Citations

Cancer Research

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 metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.


Vaccine-induced alterations in hepatic drug metabolism

May 1991

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11 Reads

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9 Citations

Vaccine

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 toxoids did not alter hepatic drug metabolism, implicating the pertussis component as the cause of the observed changes. Other pyrogenic whole cell vaccines such as typhoid and cholera also had no effect on drug metabolizing capacity. However, polyriboinosinic polyribocytidylic acid (poly I:C), a compound thought to exert its effects through the induction of interferon, induced changes comparable to those seen with DTP vaccine. The similarities in the effects following administration of DTP vaccine and poly I:C suggest that vaccine-induced alterations of drug metabolism may be mediated by immunomodulatory agents such as interferons and interleukins. Studies with purified cytokines are planned to address this question.


Citations (65)


... The activity was calculated using an extinction coefficient of 0.0436 mm⁻¹cm⁻¹. The glutathione-s-transferase activity was determined by the method of Habig et al. (1974) using 1-chloro-2,4dinitrobenzene as a substrate. Superoxide dismutase (SOD) activity was measured by the McCord and Fridovich (1969) method, based on the enzyme's catalysis of superoxide dismutation to oxygen and hydrogen peroxide, with spectrophotometric quantification. ...

Reference:

Spondias mombin L. Fruit Pulp Prevents D-Galactose-Induced Cognitive Deficits, Oxidative Stress and Neurodegeneration in Wistar Rats' Prefrontal Cortex
Glutathione S-Transferases

Journal of Biological Chemistry

... GST activity was determined following the method of Habig et al. [35]. Enzymatic activity was expressed as nanomoles of 1-chloro-2,4-dinitrobenzene conjugate formed per minute per milligram of protein. ...

Glutathione-S-transferase; the first step in mercapturic fermentation
  • Citing Article
  • January 1973

Journal of Biochemistry

... 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. ...

Bacteriophages and Plasmids in Clostridium botulinum and Clostridium tetani and Their Relationship to Production of Toxins
  • Citing Article
  • December 1989

... In glutathione peroxidase (GPX), activity from the obtained homogenates was determined by adding hydrogen peroxide (H 2 O 2 ) at 340 nm and measuring H 2 O 2 consumption for 5 min (Beutler 1975). GST activity measurement was carried out according to the method of Habig et al. (1974). GST enzyme activity in homogenates was determined by measuring at a wavelength of 340 nm for 3 min (Habig et al. 1974). ...

The human hepatic glutathione S-transferases
  • Citing Article
  • January 1976

... The ascorbate peroxidase (APX) activity was determined by monitoring ascorbate oxidation at 290 nm, using an extinction coefficient of 2.8 mM −1 cm −1 , with 1 unit defined as the oxidation of 1 nmol of ascorbate per minute (Nakano & Asada, 1981). Glutathione S-transferase (GST) activity was assessed by measuring CDNB (1-chloro-2,4-dinitrobenzene) conjugate formation at 340 nm, using an extinction coefficient of 9.6 mM −1 cm −1 , and 1 unit representing 1 nmol CDNB conjugates formed per minute (Habig & Jakoby, 1981). The peroxidase (POD) activity was determined by monitoring guaiacol oxidation at 470 nm, with 1 unit defined as an increase of 0.01 OD 470 per minute according to the method of Cakmak and Marschner (1992). ...

Assay for differentiation of Glutathione S-Transferases
  • Citing Article
  • January 1981

... A part of the rat's liver as 10 % homogenate in ice-cold sucrose and supernatant fraction after centrifugation at 600 ×g in a cold centrifuge was used for assessing the biochemical estimations while remaining part of the liver was used for histopathological studies. Antioxidant status of liver tissue was assessed from reduced Glutathione (GSH) level and enzyme activities of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione-S-Transferase (GST), and Glutathione Peroxidase (GPx) estimations [17][18][19][20][21] . The protein content in tissue homogenates was determined by the method of Lowry et al. [22] using bovine serum albumin as a standard. ...

Glutathione Transferases
  • Citing Chapter
  • December 1980

... 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). ...

Rat glutathione S-transferases: binding and physical properties
  • Citing Article
  • January 1976

... 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. ...

Tetanus toxin produces blockade of synaptic trans - mission in mouse spinal cord neurons in culture
  • Citing Article
  • January 1981