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M K Johnson

Publications and source records attributed to M K Johnson.

At least 19 recordsLinked to original sources
PubMed1992

The role of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance Raman study.

Resonance Raman spectroscopy has been used to investigate the function and properties of the iron-sulfur cluster in Escherichia coli endonuclease III. Resonance Raman spectra in the Fe-S stretching region are indicative of a [4Fe-4S]2+ cluster with complete cysteinyl sulfur coordination, and vibrational assignments are made by analogy with bacterial ferredoxins. Minor changes in the vibrational frequencies of the modes primarily involving Fe-S(Cys) stretching accompany the binding of the inhibitor thymine glycol or an oligonucleotide containing a reduced apyrimidinic site. These changes are consistent with perturbation of the orientation of the ligating cysteinyl residues and rule out the possibility that the [4Fe-4S] cluster is directly involved with substrate or inhibitor binding. It is concluded that a structural role is most likely for the [4Fe-4S] cluster in endonuclease III.

Base Sequence↗
PubMed1992

Resonance Raman and magnetic circular dichroism studies of reduced [2Fe-2S] proteins.

The structural and electronic properties of the [2Fe-2S] clusters in reduced putidaredoxin, Spinacea oleracea ferredoxin, and Clostridium pasteurianum [2Fe-2S] ferredoxin have been investigated by resonance Raman and variable temperature magnetic circular dichroism spectroscopies. Both techniques are shown to provide diagnostic fingerprints for identifying [2Fe-2S]+ clusters in more complex multicomponent metalloenzymes. The Fe-S stretching modes of oxidized and reduced putidaredoxin are assigned via 34S and D2O isotope shifts and previous normal mode calculations for adrenodoxin (Han, S., Czernuszewicz, R. S., Kimura, T., Adams, M. W. W., and Spiro, T. G. (1989) J. Am. Chem. Soc. 111, 3505-3511). The close similarity in the resonance Raman spectra of reduced [2Fe-2S] centers, in terms of both the vibrational frequencies and enhancement profiles of the Fe-S stretching modes, permits these assignments to be generalized to all clusters of this type. Modes primarily involving Fe(III)-S(Cys) stretching are identified in all three reduced [2Fe-2S] proteins, and the frequencies are rationalized in terms of the conformation of the cysteine residues ligating the Fe(III) site of the localized valence reduced cluster. D2O isotope shifts indicate few, if any, amide NH-S hydrogen bond interactions involving the cysteines ligating the Fe(III) site. Preliminary resonance Raman excitation profiles suggest assignments for the complex pattern of electronic bands that comprise the low temperature magnetic circular dichroism spectra of the reduced proteins. S----Fe(III) and Fe(II)----S charge transfer, Fe d-d, and Fe(II)----Fe(III) intervalence bands are identified.

Adrenodoxin↗
PubMed1992

Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf.

Oxidation-reduction titrations have been conducted to determine the midpoint potential (Em) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe-4S]+,0 cluster found in the enzyme, indicated the presence of a single n = 1 component with Em = -170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with Em = -180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in Em by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.

Electron Spin Resonance Spectroscopy↗
PubMed1992

Spectroscopic characterization of the alternate form of S-methylcoenzyme M reductase from Methanobacterium thermoautotrophicum (strain delta H).

Two forms (MR1 and MR2) of S-methylcoenzyme M reductase were purified from Methanobacterium thermoautotrophicum (strain delta H) as recently described (Rospert, S., Linder, D., Ellerman, J. and Thauer, R.K. (1990) Eur. J. Biochem. 194, 871-877). MR2 was at least 50-fold more active than MR1, independent of assay conditions. The two forms are spectroscopically similar, but not identical, by UV-visible, magnetic circular dichroism and resonance Raman spectroscopies. MR2 exhibited an EPR signal corresponding to 20% of the enzyme-bound nickel. Strong EPR signals similar to those previously assigned to Ni(I)F430 bound to methylreductase in Methanobacterium thermoautotrophicum (strain Marburg) (Albracht, S.P.J., Ankel-Fuchs, D., Bocher, R., Ellerman, J., Moll, J., Van der Zwann, J.W. and Thauer, R.K. (1988) Biochim. Biophys. Acta 955, 86-102) were observed in MR2-rich, log-phase, as well as in MR1-rich, slow-growing bacteria. Log-phase cells had dramatically different EPR spectra depending on whether they were removed from the fermenter (under gas flow) before or after cooling to 10 degrees C. EPR spectra of slow-growing cells were insensitive to harvesting conditions. The possible biological significance of the alternate form of methylreductase is discussed.

Circular Dichroism↗
PubMed1992

Evidence for non-cysteinyl coordination of the [2Fe-2S] cluster in Escherichia coli succinate dehydrogenase.

The consequences of replacing Cys65 in the FrdB subunit of Escherichia coli fumarate reductase by Asp or Ala have been investigated in terms of bacterial growth, enzymatic activity, and the ERP/redox properties of the [2Fe-2S] cluster. An aspartic acid residue occupies the equivalent position in E. coli succinate dehydrogenase, and the FrdBCys65Asp mutation has little effect on cell growth, enzyme activity or the physical properties of the Frd [2Fe-2S] cluster. In contrast, the [2Fe-2S] cluster was not observed in the FrdBCys65Ala mutant showing that a coordinating residue is required at this position for assembly of this cluster and significant levels of enzymatic activity. These results support the presence of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster in E. coli succinate dehydrogenase.

Cysteine↗
PubMed1992

[3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.

Amino Acid Sequence↗
PubMed1992

Age differences in using source-relevant cues.

Subjects heard words originating from 2 speakers and later decided which of the 2 speakers said the words. Older adults had difficulty with source monitoring when perceptual cues from 2 sources were similar (2 female speakers), but this difficulty was overcome when perceptual cues were distinctive (a male and a female speaker) and were the only salient cues to source. Older adults also benefited from distinctive spatial cues when these were the only salient cues to source. Older adults, however, experienced difficulties in using multiple cues (both perceptual and spatial) to source effectively, whereas younger adults were able to use multiple cues to enhance their source-monitoring performance. It is suggested that age differences in source monitoring result from differential cue utilization.

Adult↗
PubMed1992

Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

Animals↗
PubMed1992

Confirmation of the role of pneumolysin in ocular infections with Streptococcus pneumoniae.

In earlier experiments on the role of the cytolytic toxin pneumolysin in ocular infections with pneumococcus, we found that a strain carrying a deletion in the gene encoding pneumolysin was considerably less virulent than wild type when tested in an intracorneal model of keratitis in the rabbit. To confirm this result, we have constructed a strain in which pneumolysin activity was restored by transformation of the deleted strain with a plasmid bearing the complete pneumolysin gene. Hemolytic titers of pneumolysin indicated that only one copy of the plasmid per bacterium expresses the pneumolysin gene in this strain. The virulence of this strain was compared with that of wild type and deleted strains transformed with the vector lacking the pneumolysin gene. Slit lamp examination (SLE) scores for eyes infected with the restored strain were similar to those for eyes infected with wild type and significantly greater than those for the pneumolysin-deleted strain. Molecular analysis of bacteria recovered from infected corneas showed that the vector plasmid was retained; however, in most isolates of the restored strain, the plasmid underwent an excision and lost the pneumolysin gene. The cloned gene apparently persisted long enough to induce the pathologic changes, and the results confirm the importance of pneumolysin as a virulence factor in ocular infections.

Animals↗
PubMed1991

Spectroscopic evidence for a [3Fe-4S] cluster in spinach glutamate synthase.

The combination of low temperature EPR, magnetic circular dichroism, and resonance Raman spectroscopies reveals the presence of a single [3Fe-4S]+,0 center as the sole iron-sulfur prosthetic group in glutamate synthase from spinach leaves. The electronic, magnetic, and structural properties of the oxidized and reduced cluster are analogous with those of similar clusters in bacterial ferredoxins. It was not possible to convert the [3Fe-4S] cluster to a [4Fe-4S] cluster by incubating with iron under reducing conditions. Taken together with the published amino acid sequence data for plant and bacterial glutamate synthases, this suggests that the [3Fe-4S] cluster is not an isolation artifact resulting from oxidative degradation of a [4Fe-4S] cluster. The likelihood that a [3Fe-4S] cluster is an intrinsic component of all plant and bacterial glutamate synthases is discussed.

Circular Dichroism↗
PubMed1991

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase.

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.

2,2'-Dipyridyl↗
PubMed1991

Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues.

The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for the n-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose of L(-) methamidophos or of the ethyl or iso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. The n-propyl, n-butyl and n-hexyl compounds caused typical OPIDP at doses causing a peak of 70-95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be greater than 8 times the unprotected LD50 and the D-(+) isomer caused OPIDP at about 5-7 x LD50. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1991

Developmental changes in memory source monitoring.

Previous research suggests that children are more likely than adults to confuse memories of actions they imagined themselves performing with memories of actions they actually performed (Realization Judgments), but are not more likely to confuse memories of actions they had imagined performing with memories of actions they saw another person perform (Reality Monitoring). We approach these findings in terms of a theory about the processes by which people identify the sources of their recollections (Source Monitoring). This approach suggests that children may be more likely than adults to confuse memories from different sources whenever the sources are highly similar to one another. Experiments 1 and 2 tested this hypothesis by manipulating the perceptual and semantic similarity of two sources of information and testing 4- and 6-year-old and adult subjects' recollection of the sources of particular pieces of information. Experiment 3 tested the hypothesis that children are more likely than adults to mistakenly identify memories of things they imagined another person doing as memories of things they witnessed that person doing. The findings indicate that (a) people are more likely to confuse memories from similar than dissimilar sources, (b) source monitoring improves during the preschool and childhood years, and (c) children may be especially vulnerable to the effects of source similarity.

Adult↗
PubMed1991

Self-image among early adolescents: revisited.

Early adolescence is the developmental period during which children make the physical, psychological, cognitive, and social transitions to adolescence. Because the development of a healthy conceptualization of the self is foundational to adequate functioning later in life, self-image is a concept of interest to clinicians and researchers. Offer, Ostrov, and Howard (1981) conducted considerable research in the area and demonstrated that early adolescents have the capacity for self-reflection and are willing to share their reflections with others. Most early adolescents tend to be self-conscious due, chiefly, to the numerous physical and emotional changes occurring during this time period (M. P. Strommen & A. L. Strommen, 1985). Behavior is characterized by moodiness, impulsiveness, peer influence (Robin, 1985), and a growing resistance to authority. These factors usually result in an awkward child attempting to exert independence in an unprecedented fashion about a variety of topics (Manning, 1983). The purpose of this study was to investigate the self-image of early adolescents, aged 11 to 14 years, in order to plan for the health and developmental needs of middle school children and their families. Survey data were collected to answer the questions: (a) What is the self-image of a representative sample of middle school students in the community?; and (b) Are there differences in self-image based on gender, race, and parental occupation?

Adolescent↗
PubMed1991

The Stroop congruency effect is more observable under a speed strategy than an accuracy strategy.

Naming the ink color of an incongruent color word (e.g., RED printed in green) usually takes longer than naming the ink color of a color bar. However, when the ink matches the word (e.g., RED printed in red), naming tends to be faster. These phenomena are known as the STroop interference effect and the Stroop congruency effect, respectively. Although the interference effect has been robust and reliable across studies, the congruency effect tends to be elusive. It was hypothesized that this variation in outcomes might be related to subjects' response strategy. The experiment conducted to test this hypothesis induced either a speed or an accuracy strategy in two separate groups of subjects. Significant interference effects were found for both groups and the magnitudes did not differ. At the same time, the congruency effect was observed in the speed group but not in the accuracy group. These results suggest that researchers who wish to observe and study the Stroop congruency and interference effects should place special emphasis on speed. Implications of the study for a model of the Stroop effect are also discussed.

Adult↗
PubMed1990

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin.

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.

Aerobiosis↗