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Biomedical subjects

N C Robinson

Publications and source records attributed to N C Robinson.

At least 19 recordsLinked to original sources
PubMed1991

A patient-centered framework for restructuring care.

Restructuring to meet patient care needs is the greatest challenge facing nursing and hospitals today. As leaders of the largest clinical discipline, nurse executives have a professional responsibility to assist hospitals to design delivery systems that will ensure high-quality care and financial viability. The author describes how one nursing division formulated its values and beliefs into a framework for practice and operationalized the framework into a patient-centered care delivery system.

District of Columbia↗
PubMed1990

Cardiolipin-depleted bovine heart cytochrome c oxidase: binding stoichiometry and affinity for cardiolipin derivatives.

Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-[14C]acetylcardiolipin (AcCL), Kd,app less than 0.1 microM, that are not present in enzyme containing endogenous CL. An additional 2-3 lower affinity AcCL binding sites, Kd,app = 4 microM, are present in the CL-depleted complex, but these sites are also present in enzyme containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) compete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to the high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The presence or absence of hydroxyl groups in CL, MLCL, or DLCL also has little effect upon binding affinities. Binding specificity clearly favors CL since phosphatidylglycerol, phosphatidic acid, and phosphatidylcholine each have very low affinity for the CL binding sites (Kd,app greater than 20 microM). We, therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.

Animals↗
PubMed1990

Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps.

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.

Aerobiosis↗
PubMed1990

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography.

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.

Amino Acid Sequence↗
PubMed1990

Silicic acid HPLC of cardiolipin, mono- and dilysocardiolipin, and several of their chemical derivatives.

A silicic acid HPLC system in hexane-2-propanol-1 mM H3PO4 50:50:3.5 (v/v/v) is described for the analysis and/or purification of cardiolipin (CL), monolysocardiolipin (MLCL), dilysocardiolipin (DLCL), and several of their chemical derivatives. Derivatives that have been successfully analyzed include CL that is acetylated, succinylated, or tetrahydropyranylated at the 2-hydroxyl; MLCL acetylated at the 2 and 2'-hydroxyls; DLCL acetylated at the 2-hydroxyl and both 2'-hydroxyls; and MLCL tetrahydropyranylated at only the 2-hydroxyl. Water can replace 1 mM H3PO4 in the eluting solvent, but prior conditioning of the silicic acid column with the phosphoric acid solvent is necessary for acceptable chromatography. The most significant factor affecting the elution times of these compounds is the percentage of aqueous component, i.e., water or 1 mM H3PO4.

Cardiolipins↗
PubMed1988

Synthesis of cardiolipin derivatives with protection of the free hydroxyl: its application to the study of cardiolipin stimulation of cytochrome c oxidase.

Cardiolipin derivatives retaining the free hydroxyl on the polar head group were synthesized. With the use of a tetrahydropyranyl ether to protect this hydroxyl, fatty acyl substitutions were made at both of the 2-positions of cardiolipin (CL). The disubstituted derivatives were obtained in high yields. The stimulation of delipidated cytochrome c oxidase activity shows a hyperbolic dependence on the concentration of these CL derivatives. Both activation parameters, the apparent dissociation constant (Kd,app) and the maximum change in molecular activity (delta Actmax), depend on the chain length of the tails, with less dependence on the degree of saturation. Natural CL (92% C18:2, 8% C18:1) and CL disubstituted with oleic acid (47% C18:2, 52% C18:1) were equally effective at stimulating cytochrome c oxidase activity, with an apparent dissociation constant of approximately 1 microM when incubated in 0.3% Triton X-100 and assayed in lauryl maltoside. CL disubstituted with hexanoic acid, however, is a poorer activator, with an apparent dissociation constant of 6.8 microM and a delta Actmax that is 50% of that achieved with natural CL. Dilysocardiolipin, with complete removal of two of the fatty acid tails, shows negligible stimulation of cytochrome c oxidase activity.

Cardiolipins↗
PubMed1988

Subunit dissociation and protein unfolding in the bovine heart cytochrome oxidase complex induced by guanidine hydrochloride.

The response of cytochrome oxidase to the denaturant guanidine hydrochloride (Gdn.HCl) occurs in two stages. The first stage is a sharp transition centered at 1 M Gdn.HCl, whereas the second stage occurs from 3 to 7 M Gdn.HCl. In the first phase, changes occur in several spectroscopic properties: (1) the tryptophan fluorescence increases from 37% of that of N-acetyltryptophanamide to 85%; (2) the emission maximum shifts from 328 to 333 nm; (3) the circular dichroism (CD) signal at 222 nm diminishes by 30%; and (4) the Soret CD signal at 426 nm is completely abolished. These spectroscopic changes are accompanied by complete loss of the oxidase's steady-state electron-transfer activity. Of the 13 available sulfhydryl residues, 2 are reactive in the isolated enzyme, but this number increases to almost 10 in the first stage of denaturation. Subunits III, VIb, VIc, and VII dissociate from the protein complex at 0.5 M Gdn.HCl, but only subunit VII can be recovered after gel filtration chromatography [nomenclature according to Buse et al. (1985)]. In 2.5 M Gdn.HCl, the heme groups are found with a complex consisting predominantly of subunits I, II, and IV. In the second phase of denaturation, there is further disruption in the structure of the oxidase as indicated by continued decline in the ultraviolet CD signal and shift to longer wavelength of the tryptophan emission spectrum. However, the fluorescence quantum yield and number of reactive sulfhydryl groups decrease as the denaturant level is raised. Gel filtration chromatography reveals that protein and heme form a high molecular weight aggregate at 5 M Gdn.HCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1986

Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside.

Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex.

Animals↗
PubMed1986

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers.

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase.

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Sideroblastic anemia associated with thrombocytosis and a chromosome 3 abnormality.

A unique patient with sideroblastic anemia and thrombocytosis with a recurrent and ultimately fatal thromboembolic phenomenon is described. Cytogenetic analysis of bone marrow metaphases revealed a pseudodiploid chromosome complement, 46,XY,ins(3;3)(q26;q21q26). The association of thrombocytosis and ins(3;3) in patients with preleukemia or myelogenous leukemia has been reported previously. The association of ins(3;3) and thrombocytosis in our patient with sideroblastic anemia suggests that the disorder may involve a hematopoietic progenitor cell capable of giving rise to all three cell lineages. Our findings also support the suggestion that a gene on the long arm of chromosome #3 may encode a protein that, at least in part, regulates megakaryopoiesis.

Adult↗
PubMed1985

Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity.

The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron-transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers.

Animals↗
PubMed1977

Interaction of detergents with cytochrome c oxidase.

The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.

Animals↗
PubMed1976

Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species.

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.

Amino Acids↗
PubMed1975

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.

Amino Acids↗
PubMed1975

The two-domain structure of cytochrome b5 in deoxycholate solution.

The membrane protein cytochrome b5 and the polar and hydrophobic fragments into which it is cleaved by trypsin have been investigated, with major emphasis on the deoxycholate-solubilized form of the protein. Molecular weight measurements show that both the intact protein and the fragments are in a monomeric state in deoxycholate and that a small peptide of perhaps 15 residues is excised when the fragments are formed. Measurements of Stokes radius show that the major fragments are globular, but that intact cytochrome b5 has an asymmetric shape, consistent with a structure composed of two globular domains joined by a link region that may be as long as 30 to 40 A. Circular dichroism measurements were made in the far-ultraviolet and in the Soret region, and they add to previously existing data to make it virtually certain that the polar heme-containing domain is unaffected by proteolysis or by removal of deoxycholate. A significant change in the ultraviolet circular dichroism is, however, observed when proteolysis occurs and it is likely that it arises from the link between the domains, which appears to be highly structured (perhaps helical) in the intact protein, but randomly coiled after it is excised. The binding studies reported previously from this laboratory suggest that these inferences about the structure of cytochrome b5 in deoxycholate solution apply also to the protein as solubilized by detergent micelles, by phospholipid vesicles, or by the microsomal membrane.

Animals↗
PubMed1975

The binding of deoxycholate, Triton X-100, sodium dodecyl sulfate, and phosphatidylcholine vesicles to cytochrome b5.

Cytochrome b5 is composed of two domains that can be isolated after tryptic cleavage as two polypeptide fragments. One fragment is globular and hydrophilic and contains the heme; the other fragment is rich in hydrophobic amino acids and is essential for recombination of cytochrome b5 with microsomal membranes (Ito, A., and Sato, R. (1968), J. Biol. Chem. 243, 4922; Spatz, L., and Strittmatter, P. (1971), Proc. Nat. Acad. Sci. U.S. 68, 1042). Equilibrium dialysis and sedimentation equilibrium measurements of the binding of deoxycholate, Triton X-100 and dodecyl sulfate show that neither intact cytochrome b5 nor its proteolytic fragments possess high affinity binding sites for any of these amphiphiles. However, each detergent binds to the protein in a highly cooperative manner at concentrations near the critical micelle concentration. Binding measurements using the separated tryptic fragments show that deoxycholate and Triton X-100 (both nondenaturing detergents) bind to the hydrophobic fragment to the same extent as to intact cytochrome b5, and not at all to the polar fragment. Sodium dodecyl sulfate (a denaturing detergent) is bound to both tryptic fragments, but 70% of the detergent is bound to the hydrophobic fragment although it comprises only 30% of the protein mass. Less detailed measurements were made with synthetic and natural phosphatidylcholines, and show that the intact protein is quantitatively incorporated into phosphatidylcholine vesicles, but that no interaction with the polar fragment occurs. These results are interpreted in terms of the hydrophobic domain of cytochrome b5 having a diffuse hydrophobic surface that can act as a nonspecific nucleus for the formation of a micelle with a variety of amphiphilic substances. This domain of the molecule will insert into any available hydrophobic environment, whether it be detergent micelles, synthetic phospholipid vesicles, or the microsomal membrane. The incorporation of cytochrome b5 into the microsomal membrane is only a specialized case of the general property.

Binding Sites↗