The activity of gamma-glutamyl transpeptidase (gamma-GT) in populations of mononuclear cells from human peripheral blood.

The activity of gamma-glutamyl transpeptidase (EC 2.3.2.2) was observed in populations of non-stimulated mononuclear cells of human peripheral blood selected with antibody-coated magnetic particles. The highest activities of gamma-GT were found in monocytes (7.3-20.0 pkat/10(6) cells). The examination of T lymphocytes revealed a significant difference between CD4-positive (4.1 pkat/10(6) cells) and CD8-positive cells (1.2 pkat/10(6) cells). B lymphocytes and NK cells showed the lowest activities of 0.3 and 1.3 pkat/10(6) cells, respectively. The obvious differences in gamma-GT activities demonstrated here may reflect different biochemical capacities of these cell types, related to their distinct functions in the immune system.

[Biofreezer BF 80--a universally employed apparatus in preservation at very low temperatures]. / Biofreezer BF 80--eine universell einsetzbare Tieftemperaturkonservierungsapparatur.

A computer-controlled freezing apparatus is described which uses liquid nitrogen (LN2) as coolant. The freezing equipment BF 80 consists of a big cryochamber (volume 24 l), a LN2 tank and a computer for the control of the freezing procedure. The biofreezer functions in the range from 40 to -180 degrees C. A complete cooling curve can be composed of up 15 linear segments with freezing rates between 0 and 45 K.min-1. In the computer up to 80 different freezing curves can be stored and activated by simple operations. The reliability of the biofreezer BF 80 was successfully tested for the cryopreservation of lymphocytes.

Susceptibility of Rhodobacter sphaeroides to beta-lactam antibiotics: isolation and characterization of a periplasmic beta-lactamase (cephalosporinase).

Thirteen strains of the gram-negative, facultative phototrophic bacterium Rhodobacter sphaeroides were examined fro susceptibility to beta-lactam antibiotics. All strains were sensitive to the semisynthetic penicillins ampicillin, carbenicillin, oxacillin, cloxacillin, and methicillin, but 10 of the 13 strains were resistant to penicillin G, as well as a number of cephalosporins, such as cephalothin, cephapirin, and cephalosporin C. A beta-lactamase (EC 3.5.2.6) with strong cephalosporinase activity was detected in all of the resistant strains of R. sphaeroides. With strain Y-1 as a model, it was shown that the beta-lactamase was inducible by penicillin G, cephalosporin C, cephalothin, and to some minor extent, cephapirin. The beta-lactamase was located in the periplasmic space, from which it could be extracted by osmotic shock disruption. By using this fraction, the beta-lactamase was purified 34-fold to homogeneity by steps involving batch adsorption to and elution from DEAE-Sephadex A50, chromatography on Q-Sepharose, and preparative polyacrylamide gel electrophoresis. The molecular masses of the native and denatured enzymes were determined to be 38.5 kilodaltons by gel filtration and 40.5 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating a monomeric structure. The isoelectric point was estimated to be at pH 4.3. In Tris hydrochloride buffer, optimum enzyme activity was measured at pH 8.5. The beta-lactamase showed high activity in the presence of the substrates cephalothin, cephapirin, cephalosporin C, and penicillin G, for which the apparent Km values were 144, 100, 65, and 110 microM, respectively. Cephalexin, cepharidine, and cephaloridine were poor substrates. The beta-lactamase was strongly inhibited by cloxacillin and oxacillin but only slightly inhibited by phenylmethylsulfonyl fluoride or thiol reagents such as iodoacetate and p-chloromercuribenzoate.

Direct immunogold staining technique based on monoclonal antibodies: an immediate approach to the immunostaining of lymphocytes.

The report provides a procedure for labelling of monoclonal antibodies (moab's) with colloidal gold particles (mean diameter 30 or 40 nm). This procedure was exemplified for the moab's BL-T2, BL-Ig-L/1, and BL-Ia/1, which label mature T (and some populations of B) lymphocytes, B lymphocytes, and HLA class II antigen bearing lymphocytes, respectively. Each moab was characterized by an individual pH optimum for stabilization of the gold sol. The pH optima were +/- 5 (BL-T2), +/- 7 (BL-Ia/1), and +/- 9 (BL-Ig-L/1). The immunogold staining reagents were used for a direct immunostaining protocol of lymphocytes, which needs less than 100 min. The intensity of this direct immunolabelling was comparable with this one resulting from the indirect IGS. Furthermore, the comparison of the direct IGS with the immunofluorescence technique does not revealed any significant difference in the mean cell counts. The presented immunolabelling method offers a straight cell tagging, which can be advantageously used for a double staining procedure.

Immunogold cell staining using monoclonal antibodies: application to lymphoid cells on coated slides.

The immunogold staining technique was presented for light microscopic detection of cell surface antigens with monoclonal antibodies. The lymphoid cells were adhered to glass slides precoated with the adhesive poly-(dimethyl-diallyl-)ammonium chloride. The staining with the monoclonal mouse antibodies followed by the gold labelled sheep anti-mouse antibody was carried out on the cells adhered to the slides. In spite of the adherence, the patching of the gold marker as the prerequisite for the light microscopic detection occurred and the positively stained cells were visualized. On basis of this method the content of T lymphocytes (BL-T2+), B lymphocytes (BL-Ig-L/1+) and monocytes (BL-M/G+) in the mononuclear cell fraction of peripheral blood was determined in a population of healthy donors and of patients. The content of T lymphocytes of 29 healthy donors was determined using the immunogold staining technique (66.7 +/- 10.4%) and the immunofluorescence technique (67.6 +/- 9.5%). The correlation between the results obtained with both methods was highly significant (p less than 0.001).

Citrulline synthesis--a tool for investigation of the mitochondrial energy metabolism.

The conditions under which citrulline synthesis can be used to stimulate mitochondrial energy metabolism were investigated in isolated rat liver mitochondria. At 25 degrees C, with glutamate as substrate, a lag phase of citrulline formation was observed, which was neither detectable in the presence of succinate nor at 38 degrees C with both substrates. After a high-protein diet, a stationary synthesis was seen from the beginning of incubation for both substrates at either temperature. The high-protein diet was applied to reach maximal citrulline synthesis. Citrulline production was found to be enhanced twice to three times as early as after one to two days. Citrulline formation and associated oxygen uptake vs. temperature were checked in the range from 10 degrees C to 42 degrees C. A linear relationship was noted between temperature and reaction rates in the Arrhenius plot, and the share of citrulline synthesis in the total energy transformation increased with rising temperature. Citrulline synthesis was adopted in experiments to determine the equilibrium state of the adenine nucleotide translocator at 38 degrees C. Just as at 25 degrees C, the translocator operated under disequilibrium conditions at body temperature, too, and the loss of free energy was--3.2 kJ X mol-1.

Control of mitochondrial respiration.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Influence of precursors of biosyntheses on the energy metabolism of the liver cell.

The influence of variable energy demand on both the total ATP/ADP ratio and the rate of respiration was investigated in isolated hepatocytes. For hepatocytes respiring at the same rate, different ATP/ADP ratios were observed when the energy drain was caused either by ureogenesis and gluconeogenesis or by partial uncoupling. These differences were provoked by an additional effect of the precursors of syntheses studied, i.e. lactate or ornithine, on the hydrogen supply of the respiratory chain, as was reflected in terms of the reduction degree of cytochrome c in isolated mitochondria. For a fixed hydrogen supply there exists a defined characteristic ATP/ADP ratio versus rate of respiration. It is concluded that the respiratory rate, the mitochondrial redox state and the phosphorylation potential (transmitted into the extramitochondrial one by the adenine nucleotide translocator) represent a system of three interdependent parameters. The presence of metabolic precursors such as ornithine and lactate enhances the load of the cellular energy metabolism as well as the energy supply of the cell, the latter through improvement of substrate utilization. The ambivalent character of the precursors makes possible a kind of forward optimization of the hepatic energy metabolism which allows the cell to perform syntheses at a relatively high ATP/ADP ratio and, unless syntheses take place, to save energy by avoiding higher ATP/ADP ratios.

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment.

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly described the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3)--the hydrogen supply to the respiratory chain; in the resting state (State 4)--the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export--the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.

Interrelationship between oxidative energy transformation and energy consumption at mitochondrial and cellular levels.

The adaptation of oxidative energy transformation in mitochondria to the energy demand of cellular metabolism was investigated in experiments with isolated mitochondria and liver cells and by computer simulation in terms of a mathematical model. Separate draining of different energy pools allowed the determination of the relation between these pools and the elucidation of the importance of the connecting enzyme reactions to the regulation of the whole process. The following conclusions can be drawn from the results: 1. The intramitochondrial adenine nucleotide pool exhibits a homogeneous behaviour, and its changes are the signal for ATP synthesis. 2. The proton-motive force which is in near-equilibrium with the intramitochondrial phosphorylation potential is the immediate signal for the respiratory chain. 3. The intramitochondrial phosphorylation potential is transformed into the external one by a flux-dependent non-equilibrium reaction of the translocator. 4. The rate of respiration-linked ATP formation is regulated by more than one reaction step with varying control strength. 5. In both isolated mitochondria and hepatocytes an activation of respiration is provoked by a decrease in the mitochondrial energy state caused by cellular energy utilization.

Interrelationships between hydrogen-supplying reactions, respiration rate and extramitochondrial adenine nucleotide pattern.

1. The influence of a diminished hydrogen supply on the regulation of oxidative phosphorylation of isolated rat liver mitochondria in dependence on the extramitochondrial (ATP)/(ADP) ratio was investigated. 2. The hydrogen supply was diminished by using various (beta-hydroxybutyrate)/(acetoacetate) ratios as a redox buffer and the results were compared with those of experiments using perifusion of immobilized mitochondria with non-saturating substrate concentrations. 3. In both experimental approaches the influence of a diminished hydrogen pressure on the maximum (ATP)/(ADP) ratio at minimum flux was low. An extreme decrease in the (beta-hydroxybutyrate)/(acetoacetate) ratio by more than two orders of magnetitude causes the (APT)/(ADP) ratio to decrease by about 50%. 4. The load capacity of oxidative phosphorylation (maximum flux) is considerably decreased by diminished hydrogen pressure. 5. The borderline cases of purely kinetic and thermodynamic limitations of hydrogen supply were calculated by computer simulation with respect to the regulating behaviour of oxidative phosphorylation and changes in the control strength of adenine nucleotide translocator and hydrogen supply in the overall reaction. 6. A prevalent thermodynamic influence of hydrogen supply on oxidative energy transformation in the cell is discussed in the light of experimental data.

Relationship between the energy cost of ATP transport and ATP synthesis in mitochondria.

Liver mitochondria form rats kept on a high-protein diet exhibit an increased rate of respiration upon addition of ornithine in the presence of HCO2 and NH+4. This is the manifestation of intramitochondrial utilization of ATP for the synthesis of citrulline. State 3 respiration of these mitochondria could be adjusted to the same rate as that produced by ornithine by either using limiting amounts of hexokinase or titration with atractyloside. Under such conditions, in both systems the proton-motive force, the intramitochondrial ATP/ADP ratio and the redox state of the respiratory chain were the same. In contrast to this, the ATP/O ratio (equal to 2 X citrulline/O ratio) in mitochondria synthesizing citrulline was higher than the glucose 6-phosphate/O ratio in the system where ATP was trapped extramitochondrially. The ratio of these two ratios was close to 1.5 with both glutamate and succinate as respiratory substrates. From these results it can be concluded that the translocation of ATP against ADP and phosphate utilizes an amount of the chemiosmotic proton gradient equal to half of that needed for the synthesis of ATP in the inner compartment.

Influence of different energy drains on the interrelationship between the rate of respiration, proton-motive force and adenine nucleotide patterns in isolated mitochondria.

The respiration of rat liver mitochondria was stimulated by three different ways of energy drain: (a) partial uncoupling (equivalent to direct collapse of the proton-motive force), (b) intramitochondrial utilization of ATP for citrulline synthesis, and (c) extramitochondrial utilization of ATP for glucose phosphorylation. At identical rates of respiration, the intramitochondrial ATP : ADP ratios were the same in all three systems. Furthermore, the proton-motive force was the same in partially uncoupled mitochondria and in the presence of hexokinase plus glucose up to a respiration rate amounting to about 60% of that of the fully active state. However, external ATP : ADP ratios were considerably different in various systems at comparable rates of oxygen uptake, being the lowest under conditions when ATP was being utilized externally. On this basis, it is concluded that the respiratory rate is controlled directly by the proton-motive force and the mitochondrial ATP-synthesizing system operates under near-equilibrium conditions with respect to the membrane energy state parameters. However, a disequilibrium exists at the step of the transport of ATP from mitochondria to the external (cytoplasmic) compartment.

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate.

The relationship between the respiration rate and the intra- and extramitochondrial adenine nucleotides was investigated in isolated rat liver mitochondria. For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture. Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded. The results obtained for the total adenine nucleotide content (12 nmol . mg-1 protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods. Strong evidence is provided for a decrease in the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.

The multifunctional actions of beta-thujaplicin on the oxidative energy transformations as a consequence of its lipophilic and chelating properties.

The influence of the fungicidic compound beta-thujaplicin (beta-isopropyl-tropolone) on the energy transformation processes of oxidative phosphorylation was investigated in isolated rat liver mitochondria with succinate (plus rotenone) as substrate. To elucidate the observed strong inhibition of active respiration by beta-thujaplicin three possibilities were assayed: the inhibition of 1) transport processes across the inner mitochondrial membrane for inorganic phosphate, adenine nucleotides, or succinate, 2) electron flux along the respiratory chain, and 3) mitochondrial ATPase. In this respect a remarkable inhibition of both Pi transport and the translocation of adenine nucleotides could not be observed. However, the effective suppression of the DNP-induced ATPase by beta-thujaplicin explains the pronounced inhibition of active respiration. An impairment of succinate transport and the measured partial inhibition of the terminal respiratory chain at the level of cytochrome oxidase contribute to the less marked inhibition of the uncoupled respiration. The ability of beta-thujaplicin to extract mitochondrial Mg++ and the prevention of the effects of beta-thujaplicin by an excess of Mg++ in the medium suggest a common mode of action of beta-thujaplicin as a lipophilic chelator of Mg++ and other divalent cations.

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.

Functional investigations of isolated mitochondria under steady-state conditions by means of a perfusion technique.

It was observed that borosilicate glass prefilters have a high absorptive capacity for isolated rat-liver mitochondria and that this binding does not need any kind of chemical procedure. This observation has led to the development of a perifusion technique for isolated rat-liver mitochondria. During perifusion the mitochondria are immobilized on a prefilter. Their morphological and functional intactness is conserved. During the course of the perifusion no loss of marker enzymes (adenylate kinase and malate dehydrogenase) can be detected. The respiration rates in the controlled and the active state are similar to those observed in a conventional closed polarographic vessel. The respiratory control is maintained for more than 30 min. With the perifusion technique it is possible to adjust respiration rates to stationary steady states between the controlled and the active state. It was shown that the control of respiration by the extramitochondrial ATP/ADP ratio is independent of the succinate concentration in the range of 1 to 10 mM.

Control of oxidative phosphorylation by the extra-mitochondrial ATP/ADP ratio.

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate+rotenone and glutamate+malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized. The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.