Macrocompartmentation of total creatine in cardiomyocytes revisited.

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments--mitochondrial matrix space and cytoplasm--in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 microg/ml for 30 min or 600 microg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2-5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment--cytoplasm.

Reliability of sweat-testing by the Macroduct collection method combined with conductivity analysis in comparison with the classic Gibson and Cooke technique.

UNLABELLED: This study was to ascertain the reliability of sweat-testing by the Macroduct collection method combined with conductivity analysis (MCS) compared with the Gibson and Cooke technique (GCT). Sweat stimulation by pilocarpine iontophoresis was identical for both procedures, sweat being collected for 30 min on a filter paper on one forearm and in the coil of the Macroduct collector on the other. Chloride, sodium and potassium concentrations were chemically analysed both on paper-eluted and tube-collected sweat; the latter was also analysed using a conductivity analyser. Chemical analyses were compared with conductivity analyses. This prospective study was carried out on 318 subjects with MCS (118 CFs, 200 controls) and on 305 of them with the GCT (113 CFs, 192 controls). The pilocarpine iontophoresis produced adequate sweat in 96.4% of collections with GCT and in 90.9% with the MCS. Sensitivity and specificity of the Macroduct/conductivity system were comparable to the GCT. No patient detected by the GCT technique was considered negative by conductivity, but one GCT positive was "borderline" with the MCS. Six non-CF subjects identified as negative by the GCT (3.3%) were in the borderline range with the MCS. CONCLUSION: Sweat-testing by the MCS has acceptable sensitivity and specificity when performed by trained CF sweat-testing technicians. Additional studies will be required to find out if these results can be confirmed in small clinics and hospitals where testing is done infrequently. Wherever the MCS is used all positive or borderline results should be confirmed by the GCT at a reference Cystic Fibrosis Center.

Dark aerobic growth conditions induce the synthesis of a high midpoint potential cytochrome c8 in the photosynthetic bacterium Rubrivivax gelatinosus.

In several strains of the photosynthetic bacterium Rubrivivax gelatinosus, the synthesis of a high midpoint potential cytochrome is enhanced 4-6-fold in dark aerobically grown cells compared with anaerobic photosynthetic growth. This observation explains the conflicting reports in the literature concerning the cytochrome c content for this species. This cytochrome was isolated and characterized in detail from Rubrivivax gelatinosus strain IL144. The redox midpoint potential of this cytochrome is +300 mV at pH 7. Its molecular mass, 9470 kDa, and its amino acid sequence, deduced from gene sequencing, support its placement in the cytochrome c8 family. The ratio of this cytochrome to reaction center lies between 0.8 and 1 for cells of Rvi. gelatinosus grown under dark aerobic conditions. Analysis of light-induced absorption changes shows that this high-potential cytochrome c8 can act in vivo as efficient electron donor to the photooxidized high-potential heme of the Rvi. gelatinosus reaction center.

Characterization of the reaction center bound tetraheme cytochrome of Rhodocyclus tenuis.

Properties of the tetrahemic reaction center bound cytochrome have been investigated by different techniques. The mid-point potentials of the four hemes were determined by redox titration. The best fit of the data was obtained with a (n = 1) Nernst curve by using the following values of the redox parameters: Em = +420 mV for the two high-potential hemes and Em = +110 and +60 mV for the two low-potential hemes. The mid-point potentials of the two high-potential hemes are the highest reported so far. The spectral properties of the four hemes in the alpha-band have been determined by absorption spectroscopy and measurements of light-induced difference spectra in membranes of Rhodocyclus tenuis. The two high potential hemes present very similar spectra centered at 557 nm. The absorption spectra of the two low-potential hemes are very similar, and their alpha-band centered around 551 nm. Spectral properties at 100 K and the linear dichroism of optical transitions allow the determination of the relative orientations of the hemes with respect to the membrane plane. The orientation patterns thus obtained corresponds to none of the arrangements described so far for reaction center bound cytochromes.

Photoinduced cyclic electron transfer in Rhodocyclus tenuis cells: participation of HiPIP or cyt c8 depending on the ambient redox potential.

We demonstrate the participation of a cytochrome c8 and a high-potential iron-sulfur protein (HiPIP) in the photoinduced electron transfer in whole cells of Rhodocyclus tenuis depending on the redox state or background continuous illumination. At high redox potentials (above 350 mV) or under a strong background illumination (5 W m-2), the cytochrome c8 acts as the physiological electron donor to the photo-oxidized high-potential hemes of the tetraheme cytochrome bound to the reaction center. For redox potentials ranging from 200 to 310 mV or under weak background illumination (1. 25 W m-2), the electron carrier is the HiPIP. The electron transfer between cyt c8 and HiPIP and the tetraheme cytochrome has half-times of 300 and 480 micros, respectively. A slow electrogenic phase of the membrane potential is linked to their rereduction. This phase is sensitive to a specific inhibitor of the cyt bc1 complex, indicating involvement of cyt c8 and HiPIP in the photoinduced cyclic electron transfer at these two redox conditions.

In vivo participation of a high potential iron-sulfur protein as electron donor to the photochemical reaction center of Rubrivivax gelatinosus.

We have found that the only high redox potential electron transfer component in the soluble fraction of Rubrivivax gelatinosus TG-9 is a high-potential iron-sulfur protein (HiPIP). We demonstrated the participation of this HiPIP in the photoinduced electron transfer both in vivo and in vitro. First, the addition of HiPIP to purified membranes enhanced the rate of re-reduction of the photooxidized reaction center. Second, the photooxidation of HiPIP was observed in intact cells of Ru. gelatinosus TG-9 under anaerobic conditions by EPR and absorption spectroscopies. Analysis of flash-induced absorption changes showed that the equilibration of positive equivalents between the reaction center and HiPIP occurs in less than 1 ms after flash excitation. The complete re-reduction of the photooxidized reaction center is achieved in tens of milliseconds. The turnover of a cyt bc1 is also involved in this reaction, as shown by a slow electrogenic phase of the membrane potential linked to this process.