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K M Smith

Publications and source records attributed to K M Smith.

At least 163 records · Page 9Linked to original sources
PubMed1986

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions.

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.

Allosteric Regulation↗
PubMed1986

Translocation of nascent non-signal sequence protein in heated Escherichia coli.

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.

Bacterial Outer Membrane Proteins↗
PubMed1986

Bohr effect in monomeric insect haemoglobins controlled by O2 off-rate and modulated by haem-rotational disorder.

The monomeric insect (Chironomus thummi thummi) haemoglobins CTT III and CTT IV show an alkaline Bohr effect. The amplitude of the Bohr effect curve of CTT IV is about twice as large as that of CTT III. In particular, at low pH a time-dependent 'slow' decrease in p50 upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p50 (increase in affinity) correlates with the ratio of haem-rotational components exhibiting an increase of the 'myoglobin-like' haem-rotational component with high O2 affinity and high stability of the globin-haem complex. The replacement of protohaem IX by mesohaem IX and deuterohaem IX, respectively, causes an increase in O2 affinity following the order: proto less than meso less than deutero CTT Hbs. The Bohr effect, however, seems not to be affected by these porphyrin side-group substitutions. The O2 affinity is modulated by steric effects due to the substituents in position 2 and 4 via variation of the protein-haem interactions which influence the O2 release. The replacement of iron by cobalt in proto and meso CTT IV leads to an increase of the p50 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect. The natural CTT Hbs III and IV analyzed for mono-componential kinetic systems exhibit pH-dependent O2 off-rate constants: 300 s-1 (at pH 5.6) and 125 s-1 (at pH 9.7) for CTT III, and 550 s-1 (at pH 5.4) and 100 s-1 (at pH 9.0) for CTT IV. Inflection points and amplitudes of the log koff/pH plots correspond to those obtained from the Bohr effect curves indicating again a larger Bohr effect for CTT IV than for CTT III. In contrast, the O2 on-rate constants are pH-independent (kon = 1.15-1.26 X 10(8) M-1 s-1). Thus, the Bohr effect is completely controlled by the off-rate constants. Analysis for bi-componential kinetic systems employing the eigenfunction expansion method clearly identifies two kinetic components for proto-IX and deutero-IX CTT Hbs which can be attributed to the two haem-rotational components x and y (x and y differ due to an 180 degree rotation of the haem group about the alpha,gamma-meso axis; y is the myoglobin-like haem-rotational component).(ABSTRACT TRUNCATED AT 400 WORDS)

Chironomidae↗
PubMed1986

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase.

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.

Animals↗
PubMed1986

111Indium-labelled polymorphonuclear leucocyte scans in rheumatoid arthritis--an important clinical cause of false positive results.

The presence of rheumatoid arthritis in patients undergoing 111indium-labelled polymorphonuclear leucocyte scanning represents a source of possible diagnostic confusion. In such patients, the labelled cells frequently accumulate over the caecum and ascending colon, despite the absence of obvious pathology of the large bowel. The aetiology of this effect is unknown.

Abdomen↗
PubMed1986

Diurnal potassium excretory cycles in the rat.

Diurnal potassium cycles (DPC) were measured in unanesthetized undisturbed rats fed a liquid diet and maintained in a 12-h light-dark environment. A fourfold step increase in diet potassium content increased DPC amplitude without altering phase. After presentation of the high-potassium diet, the initial adaptive increase in excretion occurred within 1.5 h (diet given during dark phase) and within 6 h (diet given during light phase). On a day when food was withheld (no potassium intake), DPC were present but with a lowered amplitude. The amount of potassium excreted on a fasting day exceeded gut and extracellular fluid potassium content and was only modestly increased when rats were previously fed a high-potassium diet. In adrenalectomized rats that received no steroid replacement or received constant infusions of low levels of aldosterone, dexamethasone, or aldosterone plus dexamethasone, potassium balance and DPC were normal. It is concluded that the amplitude of DPC in the rat is determined in part by the availability of potassium from both intracellular and extracellular potassium pools; mechanisms independent of potassium intake can generate the DPC; and the presence or the cyclic secretion of adrenal steroids is not necessary for the generation of DPC in the rat.

Animals↗
PubMed1985

Proton NMR study of yellowfin tuna myoglobin in whole muscle and solution. Evidence for functional metastable protein forms involving heme orientational disorder.

Proton NMR spectra of met-aquo-myoglobin have been recorded in whole dark muscle from yellowfin tuna for the first time; in addition, spectra of the met-aquo, met-cyano, and deoxy forms were recorded in solution. The number of resolved methyl resonances in the met-aquo and met-cyano derivatives of the purified protein indicates a molecular heterogeneity, with the two species present in a ratio of approximately 3:2. These same two species, in very similar environments, are found in whole muscle. Upon reconstitution of the protein, the ratio of the two species is approximately 1:1. Specific isotope labeling in the met-cyano form reveals an interchange of 5-methyl and 8-methyl environments that is characteristic of heme orientational disorder about the alpha, gamma-meso axis. Incubation of the met-cyano protein at 37 degrees C shows that the two species are interconvertible, with the minor component declining to about one-eighth of that of the major component. This indicates that one of the two orientations is favored thermodynamically. The rate of equilibration for the met-aquo protein is exceedingly slow (half-life greater than 30 days), some 10(2) slower than for the analogous sperm whale derivative. The "reversed" heme orientation is therefore present in muscle as a kinetically trapped or metastable species, suggesting that the last step in the biosynthesis of myoglobin is similar to that of in vitro reconstitution. The presence of both heme orientations in myoglobin in whole muscle proves that heme orientational disorder is a physiological phenomenon.

Amino Acid Sequence↗
PubMed1985

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects.

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.

Animals↗
PubMed1985

1H NMR characterization of metastable and equilibrium heme orientational heterogeneity in reconstituted and native human hemoglobin.

A proton nuclear magnetic resonance study of the reaction of apohemoglobin A with both oxidized and reduced hemes reveals that at least two slowly interconverting species are initially formed, only one of which corresponds to the native proteins. Reconstitutions with isotope-labeled hemes reveal that the hyperfine-shift patterns for heme resonances in the metazido derivatives differ for the two species by interchange of heme environment characteristic of heme orientational disorder about the alpha, gamma-meso axis, as previously demonstrated for myoglobin [La Mar, G. N., Davis, N. L., Parish, D. W., & Smith, K. M. (1983) J. Mol. Biol. 168, 887-896]. Careful scrutiny of the 1H NMR spectrum of freshly prepared hemoglobin A (Hb A) reveals that characteristic resonances for the alternate heme orientation are present in both subunits, clearly demonstrating that "native" Hb A possesses an important structure heterogeneity. It is observed that this heterogeneity disappears with time for one subunit but remains unchanged in the other. This implies that a metastable disordered state in vivo involves the alpha subunit and an equilibrium disordered state both in vivo and in vitro is involved within the beta subunit. The presence of metastable disorder in fresh blood suggests an in vivo hemoglobin assembly from apoprotein and heme that is similar to the in vitro reconstitution process. The slow equilibration and known lifetimes for erythrocytes provide a rationalization for the presence of detectable metastable states. The implications of such heme disorder for Hb function are discussed.

Binding Sites↗
PubMed1985

Intermediates in the formation of the chlorophyll isocyclic ring.

Cell-free, organelle-free synthesis of Mg-2,4-divinylpheoporphyrin a(5) (MgDVP) from Mg-protoporphyrin IX monomethyl ester (Mg-Proto Me) has been described (Wong and Castelfranco 1984 Plant Physiol 75: 658-661). This system consists of plastid membrane and stromal fractions and requires O(2), NAD(P)H and S-adenosylmethionine (SAM). The synthetic 6-methyl-beta-ketopropionate analog of Mg-Proto Me was converted to MgDVP by the same catalytic system in the presence of O(2) and NADPH. SAM was not required. A compound (X) displaying the kinetic behavior of an intermediate was isolated from reaction mixtures with Mg-Proto Me as the substrate, but not with the 6-methyl-beta-ketopropionate analog as the substrate. X was identified as the 6-methyl-beta-hydroxypropionate analog of Mg-Proto Me by conversion to the dimethyl ester with CH(2)N(2) and comparison with authentic 6-beta-hydroxydimethyl ester. X was converted to MgDVP by the same catalytic system in the presence of O(2) and NADPH. We conclude that the conversion of Mg-Proto Me to MgDVP proceeds through the 6-beta-hydroxy and the 6-beta-ketopropionate esters in agreement with earlier suggestions.

Journal Article↗
PubMed1985

Cognitive therapy to achieve personal goals: results of telephone group counseling with disabled adults.

Physical disability alters a person's lifestyle and can be detrimental in its effects on mood and activity. We describe a counseling program for severely disabled persons who participated in cognitive therapy by telephone. Therapy involved solving problems related to personal dissatisfaction, loneliness, or depression. The treated group reported significantly less loneliness after treatment and were observed by their families to demonstrate more social role skills than the control group. High goal attainment reported by the treated groups was significantly correlated with decreased loneliness, positive changes in social role skills, and family expectations. Treating affective problems with scheduled telephone contact should be further evaluated because of the observed effectiveness and potential for reaching homebound clientele.

Adult↗
PubMed1984

The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity.

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.

Animals↗
PubMed1984

The specificity of biliverdin reductase. A study with different biliverdin types.

The specificity of rat liver biliverdin reductase was examined with the help of a series of synthetic biliverdins. The mixture of the four biliverdin isomers obtained by the chemical oxidation of protohemin I, protohemin XI, protohemin XIV and harderohemin were used as substrates of biliverdin reductase and were compared with the mixture of biliverdins IX alpha-delta. Biliverdin reductase (molecular form 1) from rat liver efficiently reduced the isomer mixtures of biliverdins I, XI, XIV and harderobiliverdins to the bilirubins in the presence of NADPH. The enzymatic reduction of the different biliverdin types was studied in the presence of different NADPH analogues. NADPH could be replaced by NADH, 3-acetyl NADPH and deamino-NADPH with retention of a good substrate activity only in the case of biliverdins of types I and IX and harderobiliverdins. Biliverdins XI and XIV were efficiently reduced only in the presence of NADPH and an excess of NADH. Bactobilin III-alpha was also very efficiently reduced by biliverdin reductase in the presence of both NADPH and NADH but not in the presence of the other analogues. These results indicate that biliverdin reductase reduced bilitriene acids substituted with non-polar and polar residues.

Animals↗
PubMed1984

Functional reconstitution of rat liver cytochrome P-450 with mesohemin.

After allylisopropylacetamide-mediated "suicide" destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin , a 2,4-diethyl heme-analog and result in a "meso-hemoprotein" with altered spectral but not functional characteristics.

Allylisopropylacetamide↗