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Biomedical subjects

B E Smith

Publications and source records attributed to B E Smith.

At least 145 records · Page 8Linked to original sources
PubMed1984

The inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed.

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.

Chromatography, DEAE-Cellulose↗
PubMed1984

Low-temperature magnetic-circular-dichroism spectroscopy of the iron-molybdenum cofactor and the complementary cofactor-less MoFe protein of Klebsiella pneumoniae nitrogenase.

The major metal clusters of the MoFe protein, Kpl , of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco , and of 'P' clusters in NifB - Kpl , the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl , demonstrating that the 'P' clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.

Circular Dichroism↗
PubMed1984

Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor.

When the iron-molybdenum cofactor (FeMoco) was extracted from the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae and combined with the FeMoco-deficient MoFe protein from a nifB mutant, the resultant MoFe protein exhibited the NifV phenotype, i.e. in combination with wild-type Fe protein it exhibited poor N2-fixation activity and its H2-evolution activity was inhibited by CO. These data provide strong evidence that FeMoco contains the active site of nitrogenase. The metal contents and e.p.r. properties of FeMoco from wild-type and nifV mutants of K. pneumoniae are very similar.

Binding Sites↗
PubMed1984

Continuous sciatic nerve block.

A technique of continuous sciatic nerve block is described. The method was used to relieve pain from ischaemic gangrene of the foot for 2 days before below-knee amputation and, combined with a continuous inguinal paravascular block, to provide regional anaesthesia both for the surgery and for the first 2 postoperative days.

Aged↗
PubMed1984

The effects of increased nasal airway resistance on modeled velopharyngeal orifice area estimation.

Research has shown that cleft lip and palate individuals have higher nasal airway resistance than normal subjects (Warren, Duany, and Fischer, 1969). The present work examined the predictive nature of modeled velopharyngeal orifice area calculations obtained using the hydrokinetic equation (Warren and DuBois, 1964) under conditions simulating increased degrees of nasal obstruction. The results of this project suggested that Warren's hydrokinetic method can be used to obtain accurate estimates of velopharyngeal orifice area under conditions of increased nasal airway resistance when airflow rates are nonvariant.

Air Pressure↗
PubMed1983

Nitrogenase of Klebsiella pneumoniae nifV mutants.

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.

Acetylene↗
PubMed1983

Nitrogenase from Klebsiella pneumoniae. An e.p.r. signal observed during enzyme turnover under ethylene is associated with the iron-molybdenum cofactor.

During turnover at 10 degrees C at pH 7.4 in the presence of ethylene, the MoFe protein of Klebsiella pneumoniae nitrogenase (Kp 1) exhibited an electron-paramagnetic-resonance signal with g-values at 2.12, 1.998 and 1.987. 57Fe isotopic substitution demonstrated that this signal arose from the Kp 1 FeMo-cofactor in an S = 1/2 spin state.

Coenzymes↗
PubMed1983

Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae.

The inactive MoFe protein of nitrogenase, NifB-Kp1, from two distinct nifB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) has been purified and characterized. NifB-Kp1 can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21 000. Like active wild-type Kp1, it was an alpha 2 beta 2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8 degrees C or on mild oxidation. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 +/- 0.9 Fe atoms . mol-1 and, when activated by FeMoco, had a specific activity of approx. 500 units . mg-1. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 e.p.r. signal which was probably not associated with activatable protein.

Cross Reactions↗
PubMed1983

Evidence on intramolecular electron transfer in the MoFe protein of nitrogenase from Klebsiella pneumoniae from rapid-freeze electron-paramagnetic-resonance studies of its oxidation by ferricyanide.

A transient e.p.r. signal with g-values of 2.05, 1.95 and 1.81 was observed in rapid-freezing experiments when Kpl, the MoFe protein of nitrogenase from Klebsiella pneumoniae, was oxidized by ferricyanide or by some dyes. This e.p.r. signal was assigned to the 'P'-centres and, since such signals are characteristics of [4Fe-4S]1+ clusters, provides further evidence for the 'P'-centres being in the [4Fe-4S]0 oxidation level in the dithionite-reduced protein. When 4-10-fold excesses of ferricyanide were used as oxidants, the rate of disappearance of the transient e.p.r. signal was independent of the concentrations of ferricyanide, Kpl or ferrocyanide, i.e. its disappearance was by an intramolecular process. Under some circumstances the g = 3.7 e.p.r. signal from the FeMo-cofactors disappeared at a similar rate. It was concluded that, in these circumstances, the g = 3.7 e.p.r. signal disappears, owing to intramolecular electron transfer to the 'P'-centres in the [4Fe-4S]2+ (P2+) oxidation level, whereas the gav. = 1.933 transient e.p.r. signal from the P1+ centres disappears, owing to a change in its spin state from S = 1/2 to S = 5/2 the rate of this process being maximal when there are two P1+ centres in the half-molecule. The rate of the intramolecular decay of the e.p.r. signals, 4.1 +/- 0.8 s-1, is the same as the rate of enzyme turnover. It is suggested that both processes may be linked to the same conformational change, which triggers, or is triggered by, intramolecular electron transfer.

Electron Spin Resonance Spectroscopy↗
PubMed1983

Preferences of content of psychological reports for community placement programs.

With the shift toward community-based care for mentally retarded persons has come a change in programming goals aimed more at habilitation. The information from psychological reports is frequently used as one type of information for decision making about clients although such reports have frequently been criticized for not providing relevant data. Staff members of community programs in Pennsylvania were surveyed to elicit their preferences for various types of report data. Of the categories of information included in this survey, personality dynamics information and descriptions of general emotional and personality factors were rated as less useful than was information regarding academic achievement, vocational achievement, adaptive skills, and client-examiner interaction. Implications of these findings for psychologists who assess mentally retarded clients are discussed.

Achievement↗
PubMed1983

Retrograde transport in corticospinal neurons after spinal cord transection.

Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.

Animals↗
PubMed1983

Velopharyngeal orifice area prediction during aerodynamic simulation of fricative consonants.

The present work examined the predictive nature of modeled velopharyngeal orifice area calculations obtained using the hydrokinetic equation (Warren and DuBois, 1964) during conditions simulating voiceless fricative production. Results indicated that accurate estimates of velopharyngeal orifice area can be obtained during aerodynamic events like those known to exist during fricative production. These findings were interpreted to lend support to the view that aerodynamic assessment incorporating hydrokinetic principles provides a useful, noninvasive method for clinical testing and research investigation of velopharyngeal function.

Forecasting↗
PubMed1982

Evidence on the role(s) of ATP in the mechanism of nitrogenase, from proton NMR relaxation studies on metal and nucleotide binding to the molybdenum-iron protein.

Interactions between the molybdenum-iron protein (Kp1) of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing,) EC 1.18.2.1) from Klebsiella pneumoniae and the divalent ions, Mn2+, Mg2+, Ca2+ and Ba2+, have been studied by monitoring the water proton NMR relaxation enhancement caused by the paramagnetism of Mn2+. We observed several binding sites for Mn2+, equivalent within experimental error (Kd = 209 +/- 23 microM), increasing in number from 1.0 to 2.9 per molecule in direct proportion to the specific activity of the protein. Metal binding sites on the MoFe protein are therefore essential to the enzymic function of nitrogenase. A maximum of four such sites is inferred for the fully active protein molecule. All manganese sites can alternatively bind the diamagnetic ions studied, the binding being one order-of-magnitude weaker (Kd = 2.2 +/- 0.3 mM for Mg2+; 1.6 +/- 0.2 mM for Ca2+; 3.4 +/- 0.3 mM for Ba2+), ATP and ADP form ternary complexes via Mn2+ with Kpl. The above data and other evidence on MgATP binding are discussed in terms of the site of hydrolysis of ATP during turnover and its possible bridging role between the two protein components of the enzyme.

Adenosine Triphosphate↗