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M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 73 records · Page 4Linked to original sources
PubMed1988

Nucleotide binding to uncoupling protein. Mechanism of control by protonation.

Nucleotide binding to the isolated uncoupling protein (UCP) from brown adipose tissue of hamster was studied in detail under equilibrium conditions. Besides microequilibrium dialysis and elution chromatography, a rapid anion-exchange procedure was adapted. From the concentration dependence, the KD and the binding capacity to UCP of ATP, ADP, and GTP and of the ATP analogues 5'-adenylyl imidodiphosphate (AMPPNP) and adenosine 5'-O-(3-thiotriphosphate) were determined. Elucidation of the pH dependence of nucleotide binding was the prime topic. From pH 4.6 to 7.5, the KD varies by almost 2 orders of magnitude, reaching the limits of the equilibrium methods. The pKD of GTP and ATP decreases from 6.3 to 4.3 with increasing pH. For ADP, the pKD varies only from 6.0 to 4.8. The intricate course of the pH dependence shows a "break point" of the pKD around pH 6.3, where the slope (pKD/pH) changes between about -0.2 and -1. Another break point above pH 7.2 produces a pKD/pH = -2 for ATP and GTP only. AMPPNP binding has a lower affinity (pKD about 5.8-4.1) and a pH dependence slope of -1 with no break. The breaks suggest involvement of the last ionization group (pKH approximately equal to 6.7) of the nucleotide phosphate. This agrees with the absence of a break for AMPPNP and with the shift by Mg2+ of the break for ATP to lower pH. The best-fitting model for the pH dependence requires in addition a H+ dissociating group at the binding site of UCP with a pKH approximately equal to 4, dominating the whole pH range. A second group effective above pH 7.0 amplifies the debinding specifically of ATP, not CTP or ADP. Further, the model implies binding of both NTP4- and the protonated NTPH3- or NDP3- and NDPH2- forms, however, with different affinities. On this basis, the relation between the measured overall KD and the intrinsic KD's of both nucleotide forms and the various H+ dissociation constants is derived, and the corresponding pKD/pH curves are calculated. A good fit with the data is obtained with a pKH = 3.8 for the UCP center and a pKH = 6.8 for nucleotides and with affinity ratios of 50 for NTP4-/NTPH3- and 100 for NDP3-/NDPH2-. The binding of the protonated nucleotide NTPH3- is seen only at a low pH, but with the analogue AMPPNPH3- with pKH = 7.6 it dominates the whole pH range to pH 7.2 with corresponding low affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1987

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels.

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.

Animals↗
PubMed1987

In the uncoupling protein from brown adipose tissue the C-terminus protrudes to the c-side of the membrane as shown by tryptic cleavage.

Limited proteolytic digestion of the uncoupling protein (UCP) with trypsin yielded a cleavage product only about 2 kDa smaller than the original UCP (33 kDa). This cleavage can be obtained with the solubilized isolated protein detergent micelle as well as in original brown adipose mitochondria. The cleavage site is identified by C-terminal sequence to be located near the C-terminus at lysine 292. This C-terminus, a 10 residue long peptide, is strongly hydrophilic and can be expected to be localized outside the membrane. In UCP this C-terminal stretch represents a structural difference to the similarly folded ADP/ATP carrier which does not form a corresponding cleavage product. Comparison of tryptic cleavage of UCP in mitochondria with differently broken outer membrane, in sonic particles of mitochondria, as well as in UCP proteoliposomes, indicate that the C-terminus is directed versus the cytosolic site of the membrane. Because of the easy susceptibility to trypsin, the cleavage site must be surface-exposed and the C-terminal section unusually mobile.

Adipose Tissue, Brown↗
PubMed1987

Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic alpha-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membrane-spanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.

Amino Acid Sequence↗
PubMed1986

The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate.

The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a Ki = 0.4 mM. Binding of [3H]carboxyatracylate and [3H]bongkrekate is largely inhibited by pyridoxal-P treatment with Ki approximately 1 mM. [3H]Carboxyatractylate is not and [3H]bongkrekate weakly removed by pyridoxal-P, whereas [3H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [3H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [3H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysine-containing peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier.

Animals↗
PubMed1985

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗
PubMed1985

ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance.

An unusual binding of cardiolipin to the ADP/ATP carrier has been found, which is distinguished by the relatively large amount and by the tightness of binding. High-resolution 31P NMR studies on the detergent-solubilized ADP/ATP carrier from beef heart mitochondria revealed narrow signals from phosphatidylcholine and phosphatidylethanolamine and a broadened signal of 30-40-Hz line width, suggestive of cardiolipin. Line broadening of this magnitude is to be expected when tumbling of the whole protein-detergent micelle is the only source of phosphorus spin-spin relaxation. Thus a strong immobilization of the protein-bound cardiolipin is inferred. By sucrose density gradient centrifugation phosphatidylcholine and phosphatidylethanolamine were removed, while approximately six +/- one molecules of cardiolipin remained tightly bound in the dimeric protein molecule. The cardiolipin binding was stable against treatment with sodium dodecyl sulfate although release of the inhibitor carboxyatractyloside revealed at least partial protein denaturation. Ca2+ ions did not readily interact either with the bound cardiolipin. Complete detachment of the bound phospholipid was achieved by a short heat pulse in the presence of sodium dodecyl sulfate. Denaturation of the carrier protein by guanidinium chloride or NaClO4 also led to release of the bound phospholipid. Thus different stages of protein denaturation must be envisaged.

Animals↗
PubMed1985

Interaction of fluorescent 3'-[1,5-(dimethylamino)naphthoyl]adenine nucleotides with the solubilized ADP/ATP carrier.

The binding of the 3'-[1,5-(dimethylamino)naphthoyl] (DAN) derivatives of AMP, ADP, and ATP to the solubilized ADP/ATP carrier is studied, evaluating primarily the fluorescence enhancement and 3H-labeled compound binding. DAN nucleotides also fluoresce when adsorbed to Triton X-100 micelles that are used for solubilization of the carrier. The partition of DAN-AMP between water and Triton X-100 micelles is measured, and it is shown to be shifted toward a higher content in Triton micelles with increasing salt concentration. In order to maintain a low level of fluorescence, the Triton content is decreased. The fraction of DAN nucleotide fluorescence due to carrier binding is determined by the suppression with bongkrekate (BKA). In contrast to the membrane-bound carrier, the solubilized preparation shows an increase of total BKA-sensitive fluorescence by 30-60% upon addition of ATP or ADP. In the solubilized atractylate-protein complex, the ADP-stimulated fluorescence amounts even to 80%. The suppression of fluorescence by BKA is independent of the presence of ADP or ATP, while that by carboxyatractylate (CAT) depends on ADP or ATP. The quantitation with [3H]BKA and [3H]CAT of these ligand interactions with DAN-AMP fluorescence shows that DAN-AMP fluorescence reflects the "m"-state carrier population and its redistribution under the influence of ADP or ATP. Thus, besides the "c"/"m" distribution, the kinetics of the c to m transition in the solubilized carrier also can be determined. The m share is increased to 80% when SO4, Pi, or pyrophosphate is present during solubilization. The rate of the ADP- or ATP-stimulated transition to the m state is markedly dependent on pH and on the presence of various anions, whereas the extent is little varied. The affinity decreases 4-fold going from DAN-AMP to DAN-ADP and to DAN-ATP (KD = 0.9, 1.6, and 3.2 microM). Comparison with physical binding of [3H]DAN nucleotides shows that the fluorescence yield of bound DAN-AMP is about 1.4 times higher than that of bound DAN-ATP. DAN substitution causes more than a 100-fold affinity increase for AMP and a 50-fold increase for ADP or ATP, probably because of interaction of the DAN group with a hydrophobic niche. A less specific, low-affinity displacement of DAN nucleotides by GDP, ADP, GTP and ATP (Ki = 1-2 mM) probably reflects primarily the ionic interactions at the binding center.

Adenine Nucleotides↗
PubMed1985

Immunoelectrophoretic characterization of the ADP/ATP carrier from heart, kidney, and liver.

Earlier studies gave an indication for an organ specificity of the ADP/ATP carrier. We used a modified charge-shift crossed immunoelectrophoresis for a more precise immunochemical characterization of this detergent-solubilized hydrophobic membrane protein. Immunological differences between the carrier protein from heart, kidney, and liver were demonstrated by a different electrophoretic migration of the three ligand-protein complexes in the first dimension and a distinct staining intensity, sharpness, and shape of the precipitates in the second dimension. However, the antibodies against the heart and kidney protein showed a cross-reactivity between the three antigens. The results are consistent with the view that the ADP/ATP carrier has organ-specific antigenic determinants although there is a partial identity between the carrier proteins from heart, kidney, and liver.

Animals↗
PubMed1985

The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane.

We report here, for the first time, the primary structure of uncoupling protein as established by amino acid sequencing. Like the ADP/ATP carrier, this protein has a tripartite structure comprising three similar sequences of approximately 100 residues each. These six 'repeats' exhibit striking conservation of several residues, in particular glycine and proline, at possible structurally strategic positions. Although the two proteins differ strongly in their amino acid composition, their sequences are distantly homologous. Three membrane-spanning alpha-helices can be deduced from hydropathy plots. A modified plot accounting for amphiphilic helices indicates 5-6 such alpha-segments. In addition an amphiphilic beta-strand of membrane-spanning length can be discerned. The tripartite sequence structure is also distinctly reflected in the hydropathy distribution. Based on the membrane disposition of the segments of the ADP/ATP carrier, a model for the transmembrane folding path of the polypeptide chain of the uncoupling protein is proposed.

Adipose Tissue, Brown↗