Biomedical subjects
Publications and source records attributed to W Dubiel.
Isolated rat kidney mitochondria are able to generate extraordinary amounts of adenosine. About one-third of the adenosine formed only results from the degradation of adenine nucleotides. Pyridine nucleotides may contribute to adenosine formation. Nevertheless, there must be an additional, as yet unidentified, acid-insoluble compound in mitochondria which is able to form a significant portion of adenosine.
ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1-5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.
The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.
In rat liver mitochondria there exists an AMP-dephosphorylating activity which converts external 5'-AMP to adenosine. It exhibits a pH optimum of 7.5 and a Km(AMP) of 0.085 mM. Furthermore, this activity is stimulated by magnesium (Km = 0.5 mM) and seems to be not affected by low concentrations of ATP or ADP. From the characteristics of the enzyme the existence of a 5'-nucleotidase in rat liver mitochondria which is localized on the outer surface of the inner mitochondrial membrane was concluded. The enzyme may be important for the production of cellular adenosine.
Since its discovery in 1956 the physiological function of the ATP-dependent proteolysis in reticulocytes has become an area of intensive interest. The evidence that mitochondria are the main substrate of the process during maturation of reticulocytes is summarized. The large consumption of ATP related to the degradation of mitochondria is discussed in context with possible mechanisms for ATP requirement. A novel ATP-dependent peptide release from reticulocyte mitochondria, which may be the first step in the degradation of the organelles, is demonstrated. Ubiquitin and tRNA are involved in the degradation process. Their roles, however, are not yet clear. The whole degradation cascade consisting of lipoxygenase, mitochondria susceptibility factor and ATP-dependent proteolysis, may function as a defense system against uncontrolled degradation of mitochondria in reticulocytes.
The influence of a hypobaric hypoxia on the dopamine uptake was studied in rat striatum using a concentration range of dopamine from 30 nM to 0.1 mM. Uptake kinetics were calculated by a non-linear regression computer procedure assuming one carrier protein with two binding sites and negative cooperativity. Hypoxia most prominently affected the low affinity uptake site2; a marked increase of Vmax2 was always obtained. This effect seems to be related to the degree of hypoxia and persists for 10 hours at least. For the high affinity uptake site1, a transient decrease of the Vmax1 was apparent but this effect was less reproducible than the alterations of the low affinity uptake site2. These findings are discussed in the context of increased neuronal dopamine release after oxygen deprivation both in vivo and in vitro.
The degradation of endogenous adenine nucleotides was compared in mitochondria isolated from mouse and rat liver, rat renal cortex and solid hepatoma. Mitochondria were incubated for 30 min at 37 degrees C in the presence of carboxyatractyloside and oligomycin. In rat liver mitochondria ATP, ADP and AMP were degraded by about 25% each, whereas in kidney and hepatoma mitochondria a rapid decline of ATP and ADP but no change in the AMP contents were observed. Main products formed were adenosine, inosine and hypoxanthine in all mitochondria examined. Compartment studies revealed that the main route of intramitochondrial adenine nucleotide catabolism seems to proceed via AMP dephosphorylation and subsequent adenosine deamination.
A mathematical procedure is presented which permits to calculate the steady-state concentrations of AMP, ADP and ATP in an ATP-regenerating assay containing pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. The accuracy of this procedure is demonstrated by the agreement of the calculated concentrations with the experimental data obtained in measurements of mitochondrial ATPase activities. The computer-assisted procedure can be employed (a) to determine extremely low adenine nucleotide concentrations which are difficult to obtain by direct measurements and (b) to adjust and to optimize the assay conditions according to the specific requirements of the experiment, including high concentrations of ATP and prescribed ATP/ADP ratios.
Applying a recently developed reverse-phase ion-pair HPLC technique we determined purine compounds in acid extracts of mitochondria isolated from rat and mouse liver, rat renal cortex, and hepatoma. Intramitochondrial purine nucleosides and bases were evaluated quantitatively for the first time. All mitochondria examined contain significant amounts of adenosine, guanosine, inosine, guanine, and hypoxanthine. Moreover, IMP was identified in all extracts. Adenine was detected in traces only. These results are in accordance with the concept of mitochondrial metabolism of purine compounds.
Various methods are described for the analysis of purine derivatives in biological samples by ion-pair high-performance liquid chromatography (HPLC) with both gradient and isocratic systems. A new approach is proposed that is suitable for the separation of nuclei acid constituents in different cells with a specific enzymatic activity pattern. The ion-pair HPLC methods were developed for the analysis of erythrocytes, lymphocytes and mitochondria acid-soluble fractions in clinical and experimental studies of normal and altered nucleotide metabolism. The results of studies of purine metabolite redistribution in mouse liver mitochondria during a 30-min incubation at 37 degrees C and data on purine metabolic alterations in mouse thymocytes during hepatoma growth are discussed.
The susceptibility of human serum albumin (HSA) to degradation by the ATP- and ubiquitin-dependent proteolytic system is enhanced by heat-denaturation. The steady-state levels of formed ubiquitin conjugates, however, are equal with denatured and native HSA. In contrast, both the hemin sensitive Pi release and proteolysis with native HSA are less than one fourth of that with denatured HSA. The rate limiting step for the degradation of HSA by the ATP- and ubiquitin-dependent proteolytic system must be beyond the ubiquitin conjugation steps. Differences concerning the effects of heat denaturation of mitochondria-containing stroma and HSA on ATP- and ubiquitin-dependent proteolysis are discussed.
Half-maximal rate of the ATP- and ubiquitin-dependent proteolysis of rabbit reticulocytes, determined in a cell-free system, require 0.5 mg/ml of mitochondria-containing stroma (MCS) protein at a fixed ATP concentration or 100 microM ATP at a fixed protein concentration. 20 microM hemin caused 50% inhibition of degradation of MCS.
A novel ATPase is postulated for isolated mitochondria and mitoblasts of rat liver. The enzyme is active in the presence of oligomycin and carboxyatractyloside. It can be distinguished from other well-known mitochondrial and non-mitochondrial ATPases by its insensitivity to common ATPase inhibitors and effectors and by digitonin treatment. The ATPase is localized on the outer side of the inner mitochondrial membrane. It is activated by Mg2+ in the alkaline pH range and exhibits a biphasic kinetics. The novel external ATPase of rat liver mitochondria possesses similar properties with respect to ATP-dependent protease.
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The breakdown of mitochondria-containing stroma of rabbit reticulocytes is an ATP- and ubiquitin-dependent process and there is no evidence for an ATP-dependent but ubiquitin-independent proteolysis in these cells. The ubiquitin conjugate formation with heat-denatured stroma proteins is about one-fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: an ATP-dependent and hemin-resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and a release of ubiquitin catalyzed by an isopeptidase activity.
One-hundred-and-thirteen patients with endocarditis and valvular insufficiency were studied retrospectively with special regard to indications for operation and the optimum time for cardiac valve surgery. Thirty patients (group I) had acute, 63 (group II) subacute and 20 (group III) prosthetic valve endocarditis. Group I: Eleven patients underwent surgery in the acute stage, 8 while bacteremic; 5 of the latter died perioperatively. Of the 19 patients treated medically, 16 died. Group II: All patients underwent operation in a bacteria-free state. The mortality was 5%. Group III: Eight patients had early (less than 60 days postoperatively) and 12 late endocarditis. Total mortality was 40% (71% early and 25% late mortality). Ten patients underwent reoperation, with a mortality of 20%, compared with 60% in the medically treated group. The results support the indication for early operation in acute endocarditis with progressive cardiac failure and renal failure and prosthetic valve endocarditis, even during bacteremia.
The ATP production and the main processes of ATP consumption, globin synthesis, Na+, K+-ATPase, and proteolysis of rabbit reticulocytes in two different media (1. complete medium with amino acids and glucose and 2. glucose containing electrolyte medium) were measured. The ATP formation in the complete medium amounts to 157 mmol/l X h which is 28% higher than in the incomplete medium. The increased ATP formation rate is due to increased ATP consumption by the more than doubled protein synthesis in the complete medium, whereas proteolysis and Na+,K+-ATPase are nearly unchanged. The increase in the glucose consumption in the complete medium is not fully accounted for by the increased oxidative degradation of glucose in the citric acid cycle and by lactate accumulation.
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