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Biomedical subjects

F Millett

Publications and source records attributed to F Millett.

At least 73 records · Page 4Linked to original sources
PubMed1981

Interaction between adrenodoxin and cytochrome c.

The reduction of horse heart ferricytochrome c by reduced adrenodoxin was found by stopped flow spectroscopy to follow second order kinetics with a rate constant of 7.8 X 10(6) M-1 s-1 in Tris/Cl, pH 7.5, at an ionic strength of 0.2 M, 29 degrees C. The temperature dependence of the rate constant was used to determine that the activation parameters were delta H++++ = 7.7 kcal/mol and delta S = -1.5 cal/mol degrees K. The rate constant decreased rapidly with increasing ionic strength, indicating that electrostatic interactions between the two proteins were important to the reaction. The contribution of individual lysine amino groups to the electrostatic interaction was determined by measuring the reaction rate of specifically trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives. Modification of lysines 13, 27, 72, and 79 surrounding the heme crevice decreased the reaction rate by about 2-fold, while modification of lysine amino groups in other regions of cytochrome c had decreasing effects as the distance from the heme crevice was increased. The interaction domain therefore involves specific complementary charge interactions between lysine amino groups immediately surrounding the heme crevice of cytochrome c and carboxylate groups on adrenodoxin. A semi-empirical relationship was developed for the total electrostatic interaction between the two proteins which is in quantitative agreement with both the ionic strength dependence of the reaction rate and the specific lysine modification studies.

Adrenodoxin↗
PubMed1981

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase.

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.

Animals↗
PubMed1981

Use of specific lysine modifications to identify the site of reaction between cytochrome c and ferricyanide.

The site of the reaction between horse heart ferrocytochrome c and ferricyanide was investigated by measuring the reaction rate of cytochrome c derivatives specifically modified at single lysine residues to form trifluoroacetyl or trifluoromethylphenylcarbamyl amino groups. Cytochrome c derivatives singly modified at lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice had rate constants decreased from that of native cytochrome c by factors of 1.29, 2.03, 1.12, 1.35, 1.46, 1.29, and 1.19, respectively. Modification of a given lysine with the bulky trifluoromethylphenylcarbamyl group caused nearly the same decrease in reaction rate as modification with the trifluoroacetyl group, indicating that the effect was due to removal of an electrostatic interaction between the protonated lysine amino group and ferricyanide. Modification of lysines 22, 55, 99, and 100 at the right side, bottom, and back of cytochrome c had no effect on the reaction rate. These results indicate that the reaction site is located at the exposed edge of the heme and that the electrostatic interaction between ferricyanide and cytochrome c is dominated by the lysine amino groups surrounding the heme crevice, which include lysine 86, in addition to the ones listed above. We have used the specific lysine modification results to estimate the contribution of each lysine amino group to the electrostatic interaction and have developed a semiempirical relation for the total electrostatic interaction.

Animals↗
PubMed1981

The effect of trifluoroacetyl-cytochrome c on the cytochrome c/cytochrome c oxidase reaction.

The importance of electrostatic interactions to the reaction between cytochrome c and cytochrome c oxidase is indicated most directly by the rapid increase in Km as ionic strength is increased. However, Chessa et al. (1980, Hoppe-Seyler's Z. Physiol. Chem. 361, 1077--1091) have recently found that a cytochrome c derivative trifluoroacetylated at all 19 lysine amino groups decreased the reaction rate between ferrocytochrome c and cytochrome c oxidase nearly as much as native ferricytochrome c, a known inhibitor that binds cytochrome c oxidase. They suggested that this was due to the formation of an inhibitory complex between trifluoroacetyl-cytochrome c and cytochrome c oxidase, which would argue against the importance of electrostatic interactions to the native complex. We have demonstrated that this apparent inhibition is in fact caused by a rapid electron transfer between native ferrocytochrome c and trifluoroacetyl-ferricytochrome c which decreases the concentration of native ferrocytochrome c, thus decreasing the reaction rate.

Animals↗
PubMed1980

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase.

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 greater than Lys 25 congruent to Lys 79 approximately equal to Lys 87 greater than Lys 8 approximately equal to Lys 27 approximately equal to Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.

Binding Sites↗
PubMed1980

A 19F nuclear magnetic resonance study of the interaction between cytochrome c and cytochrome c peroxidase.

The reaction between ferrocytochrome c and yeast cytochrome c peroxidase was studied using cytochrome c derivatives specifically trifluoroacetylated at single lysine amino groups. The only modifications that decreased the reaction rate were those of lysines immediately surrounding the heme crevice, lysines 13, 25, 79, and 87. Modification of lysines 22, 55, 88, and 99 had no effect on the reaction. The 19F chemical shifts of the cytochrome c derivatives trifluoroacetylated at lysines 13, 79, and 87 were not changed upon complex formation with cytochrome c peroxidase, indicating that no detectable conformational changes occurred. The cytochrome c trifluoroacetyl groups had the same T1 values in the paramagnetic fluorocytochrome c peroxidase complex as in the diamagnetic reduced form of the complex, indicating that they were more than 2.3 nm from the paramagnetic iron atom in cytochrome c peroxidase. This is consistent with a separation of at least 1.5-2.0 nm between the iron atom of cytochrome c and the iron atom of cytochrome c peroxidase.

Animals↗
PubMed1980

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase.

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.

Cytochrome c Group↗
PubMed1980

Involvement of lysines-72 and -79 in the alkaline isomerization of horse heart ferricytochrome c.

Spectrophotometric titrations of five singly modified horse heart ferricytochromes c, specifically (trifluoromethyl)phenylcarbamylated (CF3PhNHCO-) or trifluoroacetylated (CF3CO-) at lysines-13, -72, and -79, were carried out. The CF3PhNHCO-Lys-13, Lys-79, and CF3CO-Lys-79 derivatives all underwent alkaline isomerization with loss of the 695-nm band to low-spin species with an apparent pK of about 8.9, as did the unmodified cytochrome. However, modification of lysine-72 appeared to alter the reaction pathway since the CF3PhNHCO-Lys-72 derivative isomerized to a high-spin form with an apparent pK of 9.3, while the CF3CO-Lys-72 derivative isomerized to a low-spin species with an apparent pK of 9.6, indicating that lysine-72 may be the normal sixth iron ligand in the native protein alkaline isomer. These results, together with those of other workers, suggest a model for the alkaline transition in which replacement of the methionine iron ligand is dependent on a number of factors, including the local availability and relative affinities of possible ligands for the heme iron and the effects of ionic and hydrophobic interactions on the tertiary structure of the molecule.

Animals↗
PubMed1979

Interaction between cytochrome c and cytochrome b5.

The reduction of cytochrome c by cytochrome b5 was studied over a wide range of ionic strengths in four different buffer systems. The reaction rate decreased linearly as the I1/2 was increased, suggesting that electrostatic interactions are important in the interaction. The ionic strength dependence of the reaction rate was in quantitative agreement with the theory of Wherland & Gray [Wherland, S., & Gray, H.B. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 2950] only if the effective radius of the interaction was 2 A. This indicates that the interaction between the two proteins is best described as the sum of n complementary charge interactions, each involving a specific lysine on cytochrome c and a specific carboxyl group on cytochrome b5. The number of complementary charge interactions, n, was calculated to be five to seven, in agreement with the results of our specific modification studies. Ultracentrifugation and gel permeation techniques were used to demonstrate that cytochrome b5 and cytochrome c formed a stable complex at low ionic strength.

Animals↗
PubMed1977

Use of specific lysine modifications to locate the reaction site of cytochrome c with cytochrome oxidase.

The reaction of cytochrome c with trifluoromethylphenyl isocyanate was carried out under conditions which led to the modification of a small number of the 19 lysines. Extensive ion-exchange chromatography was used to separate and purify six different derivatives, each modified at a single lysine residue, lysines 8, 13, 27, 72, 79, and 100, respectively. The only modifications which affected the activity of cytochrome c with cytochrome oxidase (EC 1.9.3.1) were those of lysines immediately surrounding the heme crevice, lysines 13, 27, 72, and 79, and also lysine 8 at the top of the heme crevice. In each case, the modified cytochrome c had the same maximum velocity as that of native cytochrome c, but an increased Michaelis constant for high affinity phase of the reaction. This supports the hypothesis that the cytochrome oxidase reaction site is located in the heme crevice region, and the highly conserved lysine residues surrounding the heme crevice are important in the binding.

Binding Sites↗
PubMed1977

Effect of specific trifluoroacetylation of individual cytochrome c lysines on the reaction with cytochrome oxidase.

We have prepared three different cytochrome c derivatives, each containing a single specifically trifluoroacetylated lysine at residues 13, 55, and 99, respectively. The only modification that affected cytochrome c oxidase (EC 1.9.3.1) activity was that of lysine-13 at the top of the heme crevice. Trifluoroacetylation of lysine-13 increased the apparent Michaelis constant fivefold compared to that of native cytochrome c, but did not affect the maximum velocity. Trifluoroacetylation of lysine-55 at the left side of the cytochrome c molecule did not affect cytochrome oxidase activity in any way, nor did trifluoroacetylation of lysine-99 at the rear of the cytochrome c molecule. This indicates that the cytochrome oxidase binding site on cytochrome c involved only the front of the cytochrome c molecule and those lysines immediately surrounding the heme crevice.

Animals↗
PubMed1976

An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives.

The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might be useful probes for the interaction of cytochrome c with intact mitochondria.

Arginine↗
PubMed1976

Maximal exercise stress testing in normal and hyperlipidemic children.

Electrocardiographic and cardiovascular responses during maximal exercise were evaluated in 103 normal children and in 82 children with familial hyperlipoproteinemia. The normal and hyperlipidemic children were comparable in regards to age, weight--height index, resting and exercise blood pressures, and maximal working capacity indices. The cohort of 82 hyperlipidemic children included 61 children (29 boys and 32 girls) with well defined "monogenic" familial hyperlipoproteinemia. Segmental ST depression on the exercise electrocardiogram occurred in 8 of these 29 boys (27.6%) as compared to 4 of 55 normal boys (7.3%), P less than 0.025 and in 6 of the 32 girls (19%) as compared to 7 of 48 normal girls (14.6%), P greater than 0.1. Segmental ST depression was present in 14 of 61 (23%) children with "monogenic" hyperlipoproteinemia, as compared to 11 of 103 (10.75%) normal (x2 = 4.47, P less than 0.05). An assessment of the clinical significance of an abnormal exercise electrocardiogram in male children with "monogenic" hyperlipoproteinemia must await the following: (1) two to four decades of observation and study of the development of morbid or mortal coronary disease, or (2) the future development of improved invasive or noninvasive techniques for the early detection of covert coronary occlusive disease. Currently, maximal exercise electrocardiography cannot be contemplated as a useful indicator of eventual premature coronary artery disease in asymptomatic hyperlipidemic children.

Adolescent↗
PubMed1975

Familial hyper-alpha-lipoproteinemia: studies in eighteen kindreds.

A newly recognized familial hyperlipoproteinemia, familial hyper-alpha-lipoproteinemia, is described in 18 kindreds. Affected probands and relatives had distinctive elevations of alpha-lipoprotein cholesterol (C-HDL), slight elevations of total cholesterol, no elevation of LDL and VLDL cholesterol, and normal triglyceride levels. The proband and at least one additional first degree relative had distinctive elevations of C-HDL in 16 of 18 kindreds. Simple segregation analysis involving 84 offspring of 22 hyper-alpha X normal-alpha matings from these 16 kindreds revealed a ratio of hyper-alpha to normal of 37:47, a ratio not significantly different from 1:1 (chi 2(1) = 1.2), the ratio consistent with autosomal dominant transmission. Despite the suggestion of a major gene effect by this analysis, evaluation of the C-HDL distribution in kindred members failed to reveal bimodality, and familial correlation analysis revealed no parent-offspring correlation. The present data suggest that an environmental cause common to sibships is possibly important in causing the disorder. Longevity analysis demonstrated elongation of life expectancy for kindred members, and there was an apparent rarity of premature cardiac events.

Adolescent↗
PubMed1975

Familial hyperalphalipoproteinemia.

A 26-member kindred had the newly recognized heritable hyperlipoproteinemia, familial hyperalphalipoproteinemia. In affected family members, hyperalphalipoproteinemia was not secondary to any diseases, drugs, or industrial exposures known to elevate alpha-lipoprotein (high-density) cholesterol (C-HDL) levels. Hyperalphalipoproteinemia was transmitted vertically through three generations. There were five matings of hyperalphalipoproteinemic to normal individuals, with 25 offspring. The ratio of offspring with elevated C-HDL levels to those with normal C-HDL levels was 12:13 (0.923), a ratio not significantly different from 1 (x2 equals 0.04), the ratio predicted for an autosomal dominant trait. In affected kindred members, levels of total plasma cholesterol were slightly elevated, those of low density lipoprotein cholesterol were normal to low, those of triglyceride were normal, and those of C-HDL were consistently elevated. Affected subjects were healthy, without xanthomata, and had no unique physical or neurological features.

Adolescent↗