Inorganic pyrophosphate: formation in bacterial photophosphorylation.

Inorganic pyrophosphate is identified as the major product of photophosphorylation by isolated chromatophores from Rhodospirillum rubrum in the absence of added nucleotides.

Mechanism and evolution of the uncoupling protein of brown adipose tissue.

The uncoupling protein found in mitochondria from thermogenic brown adipose tissue is structurally very similar to two other mitochondrial carrier proteins transporting ADP/ATP and phosphate, respectively. Similarities are also seen with the mechanism of these carriers, which are part of a family of H+/OH(-)-substrate anion co-transporters, further strengthening the evidence that the uncoupling protein has evolved from this family of mitochondrial carrier proteins.

Clinical impact of a comprehensive nurse-led discharge intervention on patients being discharged home from an acute medical unit: Randomised controlled trial.

BACKGROUND: Acute medical units have increasingly been implemented in modern healthcare to ensure a fast track for treatment and care, thus increasing the number of patients being discharged. To avoid early readmissions, new approaches to discharging patients from these settings are needed. OBJECTIVE: To investigate the clinical impact of a comprehensive nurse-led discharge intervention on patients being discharged home from an acute medical unit. OUTCOMES: The primary outcome was 30-days hospital readmission. Secondary outcomes were utilisation of healthcare, including contacting emergency departments, the general practitioner or after-hours physicians; patient experience; and health-related quality of life. DESIGN: This study was a non-blinded randomised clinical controlled trial with a 1 year enrolment period from November 2014 to 2015. Group assignment was performed by computer generated codes. SETTING: The setting was a 34-bed acute medical unit at a Danish University Hospital. PARTICIPANTS: Non-surgical patients aged 18+ with more than one contact to hospitals during the last 12 months were eligible for inclusion. Furthermore, patients had to have been discharged home and had a follow-up appointment after discharge. METHODS: The intervention consisted of (1) an assessment of the patient's overall situation, (2) an assessment of their comprehension of discharge recommendations, (3) a simple discharge letter targeting the individual patient's health literacy and (4) a follow-up telephone call 2 days post-discharge. The study was carried out by a research nurse and the 1st author. Data was collected from medical records, registers and questionnaires. Intention-to-treat and per protocol analysis were performed. RESULTS: In all, 200 participants were enrolled (101 intervention; 99 control). Of these, 17 were excluded due to transfer to another hospital department and 4 did not receive the full intervention, resulting in 86 in the intervention group and 93 in the control group. At 30 days post-discharge, 22/101 (22%) in the intervention group had at least one readmission vs. 19/99 (19%) in the control group. The total number of all-cause readmissions in the follow-up period was 0.28 (SD: 0.67) in the intervention group vs. 0.26 (SD: 0.63) in the control group. There were no statistically significant differences in baseline characteristics or any of the primary and secondary outcomes. CONCLUSION: A comprehensive nurse-led discharge model focusing on the individual patient's situation and needs was not capable of reducing readmissions and healthcare utilisation. No statistically significant effects on quality of life or patients' experiences of the discharge from the acute medical unit were observed.

Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.

Kinetics of ADP, ATP transport in mitochondria as studied by the quench-flow method.

The kinetics in the millisecond range of ADP, ATP counterexchange in rat liver mitochondria were investigated using a quench-flow apparatus. The exchange was stopped with atractylate and the mitochondria were separated by centrifugation. 1. After correcting for leakage due to flow stress, apparent biphasic exchange kinetics were observed, with a more rapid phase within 100--200 ms, which extends to 10% of the total exchange. In general, the extent of the rapid phase increased when the translocation rate was changed under various conditions, in agreement with the model of the quench mechanism by atractylate. 2. The nature of the "rapid" phase was analyzed in the "steady" and "transient" state of the translocation and was shown to be caused by a delayed binding of atractylate due to competition with ADP. This quench delay results in a residual exchange which could explain the rapid part of the kinetics. 3. Deenergization of mitochondria by valinomycin or by uncoupler largely abolishes the rapid kinetic phase. This is explained by an increased availability of carrier sites at the outer face of the membrane to atractylate in the deenergized state, resulting in a more rapid quench. 4. The interpretation of the rapid exchange phase as a function of carrier sites accessible to atractylate quenching at the outer membrane face was simulated by a computer program based on the reorientating carrier model. With a set of rate constants, an approximate fit for the extent of quench delay with the experimental data is obtained.

Isolation of the ADP, ATP carrier as the carboxyatractylate . protein complex from mitochondria.

The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a blocked N-terminal. 9. From the solubilized complex (35S) carboxyatractylate can be removed by carboxyatractylate, ADP and ATP but not by ITP, etc., indicating the presence of recognizing sites specific fof ADP, ATP and therefore, identity with the ADP, ATP carrier. 10. Other reported procedures for isolating the ADP, ATP carrier are shown to either fail or have lower yield than the present, original procedure.

Uncoupling proteins: the issues from a biochemist point of view.

The functional characteristics of uncoupling proteins (UCP) are reviewed, with the main focus on the results with isolated and reconstituted proteins. UCP1 from brown adipose tissue, the paradigm of the UCP subfamily, is treated in more detail. The issues addressed are the role and mechanism of fatty acids, the nucleotide binding, the regulation by pH and the identification by mutagenesis of residues involved in these functions. The transport and regulatory functions of UCP2 and 3 are reviewed in comparison to UCP1. The inconsistencies of a proposed nucleotide insensitive H(+) transport by these UCPs as concluded from the expression in yeast and Escherichia coli are elucidated. In both expression system UCP 2 and 3 are not in or cannot be converted to a functionally native state and thus also for these UCPs a nucleotide regulated H (+) transport is postulated.

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment.

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.

A detergent-induced charge shift as a prerequisite for the electrochemical analysis of the adenine nucleotide translocator, a basic membrane protein.

The adenine nucleotide translocator is a hydrophobic, basic protein of the inner mitochondrial membrane which is solubilized by the non-ionic detergent Triton X-100. For immunochemical characterization of this membrane-protein by crossed immunoelectrophoresis a charge shift of the protein-Triton X-100 micelle by the introduction of an ionic detergent (deoxycholate) was necessary as a prerequisite to avoid unspecific precipitation of the protein. Beside the charge shift of the protein-detergent micelle, the selection, concentration and ratio of the detergents used and the choice of the agarose with different degrees of electroendosmosis should be carefully considered. The principle derived from these results provides a new methodological possibility for the immunochemical characterization of hydrophobic, basic membrane proteins.

Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential.

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.

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.

The ADP/ATP carrier from yeast (AAC-2) is uniquely suited for the assignment of the binding center by photoaffinity labeling.

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[alpha-32P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115-210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172-210 segment. 8-Azido-[alpha-32P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172-210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.

The isolation and reconstitution of the ADP/ATP carrier from wild-type Saccharomyces cerevisiae. Identification of primarily one type (AAC-2).

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

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.

Regulation of UCP3 by nucleotides is different from regulation of UCP1.

UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl- at rates equal to the reconstituted native UCP1. Cl- transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri- and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri- than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non-shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state.

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport.

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H+ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H+ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.

Some characteristics of the isolated ADP/ATP carrier.

The primary structure of the ADP/ATP carrier, comprising 297 amino acids, is discussed. Other structural features include the conformational changes related to the translocational process. Fluorescent adenine nucleotide analogs, DAN-AMP, DAN-ADP and DAN-ATP are being used for probing the transition between the "c" and "m" states. The fluorescent properties of the binding give strong support to the gated pore model, with "opposite" binding interfaces between substrate and carrier.