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1.
Protein J ; 32(1): 27-38, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23143018

ABSTRACT

The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.


Subject(s)
Electron Transport Complex IV/chemistry , Membrane Proteins/chemistry , Mitochondria/enzymology , Myocardium/enzymology , Scattering, Small Angle , Animals , Cattle , Dimerization , Lipids/chemistry , Liposomes/chemistry , Mitochondria/chemistry , Models, Molecular
2.
Ann Thorac Surg ; 88(6): 1982-8; discussion 1988, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19932272

ABSTRACT

BACKGROUND: Direct mechanical ventricular actuation (DMVA) provides non-blood contacting augmentation of ventricular function. The device has promise for supporting the pediatric heart. The purpose of this study was to assess DMVA's effect in a small animal model of heart failure. METHODS: Anesthetized rabbits (n = 6) underwent sternotomy and were instrumented for hemodynamic monitoring. A 10-MHz ultrasound probe was used for transesophageal echocardiography imaging. Heart failure (cardiac output <50% baseline) was induced with esmolol. Phenylephrine was titrated to maintain baseline mean arterial pressure. Transesophageal echocardiography imaging was acquired at baseline, heart failure, and subsequent DMVA support for 2 hours. Image analysis was used to derive ejection fraction, cardiac output, and stroke work as measures of left ventricular function. Speckle tracking software was used to derive myocardial strain rates as load-independent measures of left ventricular myocardial function. RESULTS: Mean ejection fraction was significantly increased during DMVA support (0.585 +/- 0.035) versus failure (0.215 +/- 0.014; p < 0.001). Peak global left ventricular systolic and diastolic strain rates (1/second) were significantly increased during DMVA (-2.85 +/- 0.33 and 2.92 +/- 0.37) versus failure (-1.69 +/- 0.11 and 1.99 +/- 0.14; p < 0.001 and 0.004, respectively). Peak strain rates during DMVA in the failing heart were similar to baseline. CONCLUSIONS: Direct mechanical ventricular actuation augments both systolic and diastolic left ventricular pump function. Diastolic augmentation distinguishes the device from other direct cardiac compression methods. This study demonstrated that DMVA in the small-sized, failing heart improves both systolic and diastolic myocardial function, which has favorable implications for left ventricular recovery. Direct mechanical ventricular actuation's salutary effects can be provided to the failing pediatric heart without complications of blood contact.


Subject(s)
Assisted Circulation/instrumentation , Heart Failure/therapy , Heart Ventricles , Heart-Assist Devices , Myocardial Contraction/physiology , Stroke Volume/physiology , Ventricular Function, Left/physiology , Animals , Diastole , Disease Models, Animal , Equipment Design , Heart Failure/physiopathology , Rabbits , Systole , Treatment Outcome
3.
Protein Expr Purif ; 56(2): 189-96, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17910921

ABSTRACT

Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.


Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/isolation & purification , Phospholipids/chemistry , Rhodobacter sphaeroides/enzymology , Biophysical Phenomena , Biophysics , Centrifugation, Density Gradient , Electron Transport , Electron Transport Complex IV/metabolism , Liposomes/chemistry , Phospholipids/metabolism , Protons
4.
Biochim Biophys Acta ; 1706(1-2): 126-33, 2005 Jan 07.
Article in English | MEDLINE | ID: mdl-15620373

ABSTRACT

The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s(-1) at pH 7.0 to less than 10 s(-1) at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30-50 s(-1)) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the Km values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a3 at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a3 is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O2 suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a3/CuB center prior to the reaction with O2. This step is not required for the reaction with H2O2, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O2, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.


Subject(s)
Cattle/metabolism , Electron Transport Complex IV/metabolism , Mitochondria/enzymology , Myocardium/enzymology , Potassium Channels/metabolism , Animals , Electron Transport/physiology , Heme/metabolism , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Spectrum Analysis
5.
Arch Biochem Biophys ; 416(1): 81-91, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12859984

ABSTRACT

Antibodies were raised against conserved amino acid sequences in four extramembranous portions of subunit III (sIII) from beef cytochrome c oxidase (COX) and the role of these domains in the functional activities of the enzyme was investigated. The binding of one antipeptide antibody corresponding to an externally exposed (facing the intermembrane space) domain of COX sIII (amino acids 180-189 in the primary sequence) exhibited a 30-50% stimulation of electron transfer activity in both detergent-dispersed COX and COX incorporated into phospholipid vesicles (COV). Antibody binding to two different matrix-faced domains (amino acids 57-66 and 148-159 in the sequence) resulted in small stimulations (10-25%) of COX electron transfer activity. The remaining antipeptide antibody (against amino acids 119-128) had no effect on electron transfer activity of COX in detergent solution, but exhibited a slight inhibition of activity (15%) in COV. The mechanism of antibody-induced stimulation of COX electron transfer activity was determined to be an increase in the maximum velocity of the enzyme and not due to a change in the apparent K(m) of cytochrome c interaction with COX as determined by steady state kinetic assays. Antibody binding to COX in COV increased the respiratory control ratio (an indicator of endogenous proton permeability) of COV, but had no effect on the vectorial proton pumping activity of COV. These results suggest that these conserved, hydrophilic domains of COX sIII are conformationally linked to the electron transfer function of the enzyme in subunits I and II and that sIII may serve as a regulatory subunit for COX electron transfer and proton pumping activities.


Subject(s)
Antibodies/pharmacology , Antibody Specificity , Electron Transport Complex IV/immunology , Electron Transport Complex IV/metabolism , Myocardium/enzymology , Amino Acid Sequence , Animals , Binding Sites , Cattle , Detergents/chemistry , Electron Transport , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/drug effects , Enzyme Activation/drug effects , Molecular Sequence Data , Phospholipids/chemistry , Phospholipids/metabolism , Protein Conformation , Protein Structure, Tertiary/physiology , Protein Subunits/chemistry , Protein Subunits/drug effects , Protein Subunits/immunology , Protein Subunits/metabolism , Protons
6.
Protein Expr Purif ; 26(1): 122-30, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12356479

ABSTRACT

Liposomes containing bovine heart cytochrome c oxidase (COV) prepared by the cholate dialysis technique were purified from those devoid of the enzyme using discontinuous sucrose density ultra centrifugation to eliminate interference in proton-pumping assays. This technique was also used to purify liposomes containing cytochrome c oxidase depleted in subunit III (COV-III), a COX enzyme preparation with altered subunit structure, to assess if the technique could be applied to COX enzymes in which structural and functional changes have occurred. Upon discontinuous sucrose density ultra gradient ultracentrifugation, either COV or COV-III were separated into two bands. Liposomes devoid of enzyme sedimented into the 12% sucrose layer, whereas enzyme-containing liposomes (pCOV or pCOV-III) were found in the 13% sucrose layer. The yield of both pCOV or pCOV-III was greater than 60% (based on heme aa(3) content), suggesting a similar distribution of cytochrome c oxidase (COX) and subunit III-depleted enzyme (COX-III) in the purified liposomes. The number of COX or COX-III molecules per phospholipid vesicle in purified fractions was estimated to be two. Removal of subunit III (M(r)=29,918) from COX resulted in a 30% decrease in electron transfer activity (either in COV-III or pCOV-III) when compared with COV and pCOV, respectively. Both pCOV and pCOV-III exhibited low endogenous proton permeability, as assessed by possessing high respiratory control ratios (14 and greater) and by having similar valinomycin concentration dependencies for stimulation of electron transfer activity in the presence of saturating amounts of CCCP. COV-III and pCOV-III exhibited a 39-44% decrease in proton-pumping activity when compared with COV and pCOV. These results showed that the separation of COX containing liposomes from those lacking enzyme by sucrose density gradient centrifugation can be used to characterize the biophysical properties of these liposomes.


Subject(s)
Electron Transport Complex IV/isolation & purification , Electron Transport Complex IV/metabolism , Liposomes/chemistry , Phospholipids/chemistry , Animals , Cattle , Centrifugation, Density Gradient , Electron Transport , Electron Transport Complex IV/chemistry , Liposomes/isolation & purification , Mitochondria, Heart/enzymology , Protons , Sucrose
7.
J Nutr Biochem ; 6(7): 385-391, 1995 Jul.
Article in English | MEDLINE | ID: mdl-12050000

ABSTRACT

The relative quantities of cardiac laminin, fibronectin, cytochrome c oxidase (CCO), and isomyosin types were studied by gel electrophoresis in male rats fed copper-deficient diets beginning either from the time of weaning for 5 weeks or from 5 weeks postweaning for 6 weeks with one group of copper-repleted rats. Increased levels of fibronectin and V(3) isomyosin but decreased levels of CCO subunit IV and laminin were found in weanling copper-depleted rats. In contrast, postweanling copper-depleted rats exhibited only increased levels of fibronectin and decreased levels of cardiac CCO subunit IV. Repletion of copper-deficient rats for 6 weeks was not sufficient to restore CCO subunit IV to the same level as controls. These results confirm that biochemical lesions in the basal laminae are a result of copper restriction. The decreased nuclear encoded subunits of CCO may help explain some of the mitochondrial pathology observed in dietary copper restriction. Increased V(3) isomyosin levels with low ATPase activity may help to conserve to a limited extent the ATP levels in copper-deficient cardiac tissue. These protein changes are consistent with the known morphological alterations of hearts from copper-restricted rats.

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