Binding of Ca2+ to glycoprotein from bovine heart mitochondria

It is shown that glycoprotein from bovine heart mitochondria which forms Ca2+-selective conductance channels in a bilayer lipid membrane possesses Ca2+-binding activity. Ca2+-binding sites of two kinds were revealed in the glycoprotein molecule: high affinity sites with Kd = 2.8 X 10(-6) M and low affinity sites with Kd 1.1 X 10(-5) M. Ca2+-binding by the high affinity sites occurs co-operatively. The Hill coefficient is about 2.     

Association of ferrochelatase with Complex I in bovine heart mitochondria

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor. Immunoblot experiments confirmed the presence of ferrochelatase in Complex I, but not in Complexes II or III. Some phospholipids, including phosphatidylserine and cardiolipin, stimulated NADH-dependent heme synthesis from ferric ion. When purified ferrochelatase was incubated with the low molecular weight form of NADH dehydrogenase prepared from Complex I, heme synthesis from ferric ion occurred by the addition of NADH. FMN markedly elevated the synthesis. These results indicate that ferrous ion is produced by NADH oxidation in Complex I and is then utilized for heme synthesis by ferrochelatase.     

Binding of dicyclohexylcarbodiimide to a native F1-ATPase-inhibitor protein complex isolated from bovine heart mitochondria

The effect and the binding of dicyclohexylcarbodiimide (DCCD) to a soluble native F1-ATPase-inhibitor protein complex (F1-IP) isolated from heart mitochondria was studied. About one mol DCCD bound per mol F1-IP complex; this inhibited its ATPase activity by more than 95%, ever under conditions that led to maximal hydrolysis. Bound DCCD localized to beta-subunits of the F1-IP complex. Cross-linking of the DCCD labeled complex with N-(ethoxy-carbonyl)-2-ethoxydihydroquinoline yielded a protein with a Mr 65,000-67,000 that contained IP as evidenced by its reaction with IP antibodies. No alpha-subunits were detected in this cross-linked product. The Mr 65,000-67,000 protein corresponds to beta-subunits cross-linked with IP (Klein et al, Biochemistry 1980; 19, 2919-2925). However, no DCCD was found in the cross-linked beta-subunit-IP product of labeled native F1-IP. Thus the beta-subunit in contact with IP is distinct from the other two beta-subunits of the enzyme.     

The phosphorylation of subunits of complex I from bovine heart mitochondria

In bovine heart mitochondria and in submitochondrial particles, membrane-associated proteins with apparent molecular masses of 18 and 10 kDa become strongly radiolabeled by [(32)P]ATP in a cAMP-dependent manner. The 18-kDa phosphorylated protein is subunit ESSS from complex I and not as previously reported the 18 k subunit (with the N-terminal sequence AQDQ). The phosphorylated residue in subunit ESSS is serine 20. In the 10 kDa band, the complex I subunit MWFE was phosphorylated on serine 55. In the presence of protein kinase A and cAMP, the same subunits of purified complex I were phosphorylated by [(32)P]ATP at the same sites. Subunits ESSS and MWFE both contribute to the membrane arm of complex I. Each has a single hydrophobic region probably folded into a membrane spanning alpha-helix. It is likely that the phosphorylation site of subunit ESSS lies in the mitochondrial matrix and that the site in subunit MWFE is in the intermembrane space. Subunit ESSS has no known role, but subunit MWFE is required for assembly into complex I of seven hydrophobic subunits encoded in the mitochondrial genome. The possible effects of phosphorylation of these subunits on the activity and/or the assembly of complex I remain to be explored.     

The nuclear encoded subunits of complex I from bovine heart mitochondria

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, Iα, Iβ and Iλ, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.     

The nuclear encoded subunits of complex I from bovine heart mitochondria

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, I alpha, I beta and I lambda, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.     

Analysis of the subunit composition of complex I from bovine heart mitochondria

Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Ilambda represents the extrinsic arm, subcomplex Ialpha consists of subcomplex Ilambda plus part of the membrane arm, and subcomplex Ibeta is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Ilambda, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex Ibeta, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566+/-2 Da) also detected in the HPLC analysis of subcomplex Ibeta. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa.     

Binding of creatine kinase to heart and liver mitochondria in vitro

Phosphate extraction of heart mitochondria results in the release of creatine kinase. Under appropriate conditions phosphate-extracted mitochondria are able to rebind the creatine kinase, either from crude extracts or as the purified enzyme. Heart mitochondria are able to bind up to sevenfold more creatine kinase than they originally contained. The association is specific since the cytoplasmic isozyme from heart (MM) does not bind, and does not interfere with the binding of the mitochondrial isozyme even when MM is present in large excess. It is interesting that although liver mitochondria do not contain the mitochondrial isozyme of creatine kinase they are able to bind approximately the same amount of the enzyme as the heart mitochondria.     

The binding of copper ions to copper-free bovine superoxide dismutase. Kinetic aspects.

The kinetics of reconstitution of bovine superoxide dismutase from Cu2+ and the copper-free enzyme have been studied by activity, u.v.-absorption, electron-paramagnetic-resonance and pulsed-nuclear-magnetic-resonance measurements. The process appears to be first-order up to 80% completion in most conditions, and is pH-dependent, with an apparent pK of 6.5. U.v.-absorption and solvent proton relaxation rate measurements show that fast binding of Cu2+ occurs, and the initial ligands are likely to be, at least in part, those of the native active site. The recovery of the native activity and spectroscopic properties is a slow process with activation energies of 92 kJ/mol at pH 5.3 and 8.4kJ/mol at pH 8.1 and can be described as a rearrangement of the site around the bound metal. The rate of this process is lower in partially recombined protein samples, probably because of intersubunit interactions.     

Binding of divalent copper and calcium ions to poly I

The effect ot Cu²⁺ and Ca²⁺ ions, on the ultraviolet differential (UVD) spectra of single-stranded poly I was studied and the coordination (Δε_b) and conformation (Δε_c) conponents of the spectra calculated The comparison of Δε_b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) ot inosine-5'-monophosphate (IMP) is a coordinating site tor Ca²⁺ and Cu²⁺ ions on the polymer bases. The binding ot Ca²⁺ and Cu²⁺ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) × 10³ cm⁻¹ The calculation of concentration dependencies tor the association constants (K“) ot Ca²⁺ and Cu²⁺ ions binding to poly I bases shows that the binding is cooperative The K“ values for the poly I + Ca²⁺ complex are two orders of magnitude lower than those for the poly 1 + Cu²⁺ complex At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I At Ca²⁺ concentrations exceeding 10⁻³ M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

The post-translational modifications of the nuclear encoded subunits of complex I from bovine heart mitochondria

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-alpha-acetylated, and a 14th, subunit B18, is N-alpha-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-alpha-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.     

Binding of spermidine and putrescine to energized liver mitochondria.

The binding of spermidine and putrescine to mitochondrial membranes was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes (V. Di Noto, L. Dalla Via, A. Toninello, and M. Vidali Macromol. Theory Simul. 5, 165-181, 1996). Results demonstrate the presence of two monocoordinated binding sites (S1 and S2) for spermidine and one monocoordinated binding site (S2) for putrescine, all exhibiting high capacity and low affinity. It is proposed that differences in the polyamines' flexibility and hydrophilicity perhaps contributes to the observed variations in their interactions with the two sites. A comparison of the binding parameters of these polyamines with those of spermine reveals differences in the specific function of the S1 and S2 sites, identified in studies of spermine binding (L. Dalla Via, V. Di Noto, D. Siliprandi, and A. Toninello Biochim. Biophys. Acta 1284, 247-252, 1996).     

Preparation and characterization of homogeneous coupling factor 6 from bovine heart mitochondria.

Coupling factor 6 (F₆) and mitochondrial ATPase inhibitor were isolated from the rutamycin-sensitive ATPase complex of bovine heart mitochondria by heating and fractionation with ethanol. F₆ appeared in acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and urea as a single band corresponding to a molecular weight of 8,000. This protein which is required for the ³²P_i-ATP exchange in submitochondrial particles treated with silicotungstate was very sensitive to trypsin.     

Partial purification and characterization of the phosphate transporter from bovine heart mitochondria.

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the Pi/OH exchange, but not the Pi/Pi exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K+. Both Pi/OH and Pi/Pi exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.