Biomedical subjects
Publications and source records attributed to M K Johnson.
Pneumolysin-negative mutants of Streptococcus pneumoniae were isolated after mutagenesis with ethyl methane sulfonate. Though totally devoid of pneumolysin, these strains produced alpha hemolysis on blood agar when incubated aerobically. It is concluded that the alpha hemolysis typical of pneumococci is unrelated to their pneumolysin content.
The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.
The resonance Raman spectra of ferredoxins (Fd) I and II from Desulfovibrio gigas are reported using 4579 A Ar+ laser excitation. The (3Fe-3S) center in Fd II has a characteristic resonance Raman spectrum, readily distinguishable from those of (2Fe-2S) or (4Fe-4S) clusters. Reduction of Fd II produces a marked alteration in the resonance Raman spectrum. Fd I is shown to contain both (3Fe-3S) and (4Fe-4S) Fd-type clusters. The results illustrate the potential of resonance Raman spectroscopy in Fe-S cluster identification, even in cases where more than one cluster type is present.
Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.
The reaction of bovine erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) with a set of structurally related phosphorothiolates was studied in order to investigate the properties of the phosphorylated enzymes and to identify the leaving group. OOS- and OOS-trimethyl phosphorothiolates and their triethyl analogues inhibit acetylcholinesterase reversibly and by progressive inhibition, and the phosphorylated enzymes undergo both spontaneous reactivation and aging. For each compound the enzyme-inhibitor dissociation constant, and the rate constants for inhibition (ka), reactivation and aging have been derived. The OOS-compounds are more potent inhibitors than the OOS-compounds, and the derived inhibited enzymes reactivate and age faster. By comparing reactivation and aging rate constants with those obtained from phosphorylated enzymes of known structure it was concluded that the leaving group of during phosphorylation is the S-alkyl. SSS-trimethyl and -triethyl phosphorothiolates also form reversible complexes and inhibit the enzyme progressively. With these inhibitors the phosphorylated enzymes did not reactivate either spontaneously or in response to oximes under conditions successful for the other inhibitors. The ka values (37 degrees C, pH 7.4) range from 30 M-1 X min-1 (OOS-trimethyl phosphorothiolate) to 6.7 X 10(3) M-1 X min-1 (OOS-triethyl phosphorothiolate) as compared to 1.25 X 10(5) M-1 X min-1 determined for isomalathion (O, S-dimethyl S-(1,2-dicarbethoxyethyl)-phoshporodithioate), which was used as one of the reference compounds. If the inhibitory potency of the trialkyl phosphorothiolates is calculated from measurements made after a fixed preincubation time the results in ka values will be misleading.
M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.
Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.
The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.
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Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d1 with an affinity constant, Kaff = 360 M-1, whereas imidazole binds to both hemes c and d1 with kaff = 35 and 55 M-1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d1 are low-spin ferrihemes in both derivatives, although the epr of the heme d1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivatives in which heme d1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.
Heme d1 has been extracted from Pseudomonas nitrite reductase. Imidazole, cyanide, and chloride-ferroheme, and CO, NO, cyanide, imidazole, and pyridine-ferroheme complexes have been prepared for study by UV/vis spectroscopy, and in some cass by epr and low-temperature mcd as well. Iron determinations have been carried out to assess extinction coefficients. Absorption spectra were used to monitor the transition of chloride-ferriheme d1 to an alkaline form of ferriheme d1 and a pka of 6.5 was determined for the process. The epr spectrum of chloride-ferriheme possessed the characteristic g = 6 signal of high spin (S = 5/2) iron, but the alkaline-ferriheme form gave no detectable epr signals. Electron paramagnetic resonance spectra were also obtained for cyanide and imidazole-ferriheme d1 and for NO-ferroheme d1. The imidazole complex gave signals that were very weak in comparison with the cyanide complex, but mcd measurements of imidazole-ferriheme d1 were consistent with it being a low-spin (S = 1/2) system. The epr signals of NO-ferroheme d1 were similar to those of the corresponding holo-enzyme complex. Reduction of alkaline-ferriheme d1 was found to be affected by the presence of oxygen, but under N2 give the same result with ascorbate and dithionite. Autoreduction of alkaline-ferriheme d1 was observed when placed under CO, and NO, atmospheres, or when treated with pyridine.
Many, but not all, reports of delayed neuropathy associated with acute poisoning by trichlorphon refer to cases in U.S.S.R. Adulteration of technical trichlorphon with the ethyl analogue would greatly increase the neurotoxic hazard but analysis of a few samples has not revealed such impurities. Simultaneous ingestion of alcohol does not appear to increase neuropathic hazard. In hens double doses of trichlorphon each exceeding unprotected LD50 can produce moderate neuropathy associated with appropriately high inhibitions of neurotoxic esterase. Similar results are obtained with 2 doses of 10 x LD50 of dichlorvos. In vitro the inhibitory power of dichlorvos against neurotoxic esterase of hen brain is 0.02 x the power against acetylcholinesterase. This ratio correlates reasonably with the ratio of LD50/neuropathic dose. The factor for human brain enzymes is 0.06 suggesting that man is more susceptible to neuropathic effects of near-lethal doses of circulating dichlorvos. It is concluded that the only neuropathic hazard to man from good quality trichlorphon arises from rapid ingestion of massive doses. To obtain critical levels of inhibition of neurotoxic esterase and to cause neuropathy in man by repeated doses would require each dose to be severely toxic. Dichlorvos ingested in large doses is likely to kill rather than to cause neuropathy.
Pneumolysin was bound by human polymorphonuclear leukocytes in a reaction which occurred very rapidly at 0 degrees C. Low concentrations of pneumolysin were found to stimulate leukocyte migration and lysosomal enzyme secretion. At increasing lysin levels, inhibition of spontaneous migration and chemotaxis, cell death, and lysis were observed. Pneumolysin was also found to lyse platelets and to activate serum to become chemotactic.
A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.
The magnetic-circular-dichroism (m.c.d.) spectra of methymyoglobin cyanide and oxidized horse heart cytochrome c were measured in the region of the Soret band over a range of temperatures from 1.5 to 50 K and in fields from 0 to 5T. A similar study has been made with reduced bovine heart cytochrome c oxidase, which contains one high-spin ferrous haem, namely a3. M.c.d. magnetization curves characteristic of an isolated Kramer's ground state with spin S = 1/2. These curves contrast with the magnetization curve of the high-spin ferrous haem with spin S = 2. The electronic ground state of the latter compound contains zero-field components that are thermally accessible over the temperature range of the experiment. Hence the magnetization curves are a complex nested set. The magnetization curves of the S = 1/2 proteins were analysed and it is shown that it is possible to make estimates of the ground-state g-factors even in the presence of rhombic anisotropy, provided that some knowledge of the polarizations of the electronic transitions is available. The striking difference between the m.c.d. magnetization curves of a simple S = 1/2 paramagnet and magnetically complex ground state should prove extremely useful when m.c.d. spectroscopy is sued to probe the magentic properties of metal centres in proteins, and should have wide application beyond the field of haemoproteins.
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The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.
1. It was proposed [Johnson (1974) J. Neurochem. 23, 785--789] that both inhibition of neurotoxic esterase of nervous tissue and subsequent 'aging' of the inhibited esterase are necessary events in the pathogenesis of organophosphate-induced delayed neuropathy: aging has now been demonstrated with a number of neurotoxic compounds. 2. Reactivation by KF was observed for hen brain neurotoxic esterase inhibited by 14 organophosphates and phosphonates, and time-dependent loss of reactivatibility (aging) occurred in every case. 3. For five other compounds no reactivation occurred and aging could not therefore be established, but independent evidence for two compounds suggests that aging was rapid. 4. Half-lives of aging of neurotoxic esterase inhibited by phosphates ranged from less than 1 min to 10 min, and for phosphonates the range was 3--600 min. 5. The relationship of these findings to the mechanism of toxicity and to the prospects of therapy are considered. 6. Aging occurred rapidly with aryloxy and linear alkoxy groups attached to phosphorus and slowly with a highly branched alkoxy substituent: these effects seem incompatible with an SN1 (dealkylation) mechanism.