Spinach chloroplast ATP-dependent endopeptidase: Ti-like protease.

The soluble fraction of spinach chloroplast was used for purification and characterization of an ATP-dependent protease. Purification included Q Sepharose Fast Flow, hydroxylapatite and FPLC Superose 6 column chromatography. The isolated enzyme requires ATP and Mg2+ for stimulation and represents a ubiquitin independent serine protease, containing essential sulphydryl group(s). By using fluorogenic peptides a similarity of chloroplast protease to Escherichia coli Ti protease was observed. The chloroplast protease is immunochemically cross-reactive with the bacterial protease Ti.

Yeast mitochondrial ATP-dependent protease: purification and comparison with the homologous rat enzyme and the bacterial ATP-dependent protease La.

Homogenous ATP-dependent protease has been isolated for the first time from mitochondria of yeast Saccharomyces cerevisiae. The enzyme molecule consists of six 120 kDa subunits. It is a serine protease with an absolute ATP requirement for its activity. Basic enzymatic characteristics of the yeast protease are similar to those of the corresponding rat mitochondrial enzyme and of the E. coli protease La. The yeast enzyme immunochemically cross-reacts with the bacterial protease La.

Low translational efficiency of the F1-ATPase beta-subunit mRNA largely accounts for the decreased ATPase content in brown adipose tissue mitochondria.

The half-life of the F1-ATPase beta-subunit (F1-beta) mRNA in ATPase-poor brown adipose tissue (BAT) (t1/2 = 9.5 h) was found to be 3-7-fold shorter than in liver (t1/2 = 27 h) and heart (t1/2 = 63 h) of mice. When translated in reticulocyte lysate, a 2-3-fold lower efficiency appeared with F1-beta mRNA from BAT than from other tissues. The in vitro synthesized F1-beta protein precursors of BAT, liver and heart origin were imported and processed by mouse liver mitochondria with equal efficiency. The results indicate that the pool of abundant F1-beta mRNA in BAT is not fully translatable, most likely due to its low metabolic stability.

Increased steady-state levels of several mitochondrial and nuclear gene transcripts in rat hepatoma with a low content of mitochondria.

Cells from a rapidly growing rat Zajdela hepatoma were shown to contain (on a protein basis) five-times less mitochondria than hepatocytes from resting or regenerating rat liver. Transcripts of four nuclear genes for representative mitochondrial membrane proteins (beta-F1 subunit and N,N'-dicyclohexyl-carbodiimide-binding protein of ATP synthase, subunit IV of cytochrome oxidase and ADP/ATP translocase) were present in 2-4 times higher amounts in the poly(A)-rich RNA of the hepatoma than in the corresponding RNA fraction from resting or regenerating rat liver. The liver and hepatoma transcripts for the beta-F1 subunit were translated in an in-vitro system with equal efficiency. Pulse-chase labeling of isolated Zajdela hepatoma cells and hepatocytes from resting and regenerating liver revealed a relative excess of the newly synthesized beta-F1 subunit in the tumor cells. The half-life of the beta-F1 subunit was significantly shorter in the hepatoma cells than in hepatocytes from resting and regenerating liver. The contents of transcripts of three mitochondrial genes examined (cytochrome oxidase subunits I and II and NADH-ubiquinone reductase subunit 2) in Zajdela hepatoma mitochondria were about five-times higher than in the mitochondria of the resting cells and 3-4 times higher than in the organelles of the regenerating organ. The results indicate that events other than transcription (most likely post-translational) may be responsible for the reduced content of mitochondria in tumor cells.

Changes in nuclear protein pattern by glucocorticoid treatment of lymphoid cells.

Glucocorticoids initiate a cytolytic process in lymphoid cells. The ultimate response is preceded by several phenomena. It is generally accepted that these are mediated through messenger proteins. The induction of these proteins is considered the primary effect of glucocorticoids. However, as yet specific gene products have not been identified. In electrophoretic assays, we observed an increased concentration of 6 nuclear proteins within a few hours of exposure of lymphoid cells to glucocorticoids. These proteins displayed prominent DNase activity. However, further studies showed: (1) that the proteins concerned are histones, (2) that histones are more easily extracted after glucocorticoid-induced alterations of lymphoid cells, and (3) that basic proteins in general express nuclease activity under certain experimental conditions. This nuclease activity is, however, artifactual. Therefore, though the changes observed are certainly related to glucocorticoid-induced effects, these do not reflect the induction of specific proteins. The results of the study indicate that glucocorticoid-induced changes in the concentration of cellular proteins should be interpreted with caution.

Control of mitochondrial transcription by thyroid hormone.

Thyroid hormone regulation of rat liver mitochondrial transcription was investigated. Steady-state levels of mitochondrial transcripts were measured by Northern blot analysis using cloned fragments of rat mtDNA. Thyroid hormone increased the steady-state concentrations of all mitochondrial mRNAs by 2-8 fold after 1-3 days of hormone treatment, whereas no significant change in the mitochondrial rRNA was observed. Analysis of transcript synthesis in isolated mitochondria shows that part or all of this increase is accounted for by elevated synthesis. Mechanisms by which thyroid hormone regulates transcription of the mitochondrial genome are discussed.

Inhibition of mitochondrial protein synthesis in regenerating rat liver stimulates mitochondrial transcription.

Partially hepatectomized rats were treated in vivo with thiamphenicol for 3 days to block mitochondrial protein synthesis. Protein synthesis, RNA synthesis and the steady-state levels of individual transcripts were measured in mitochondria in vitro in the absence of thiamphenicol. Incorporation of [35S]methionine and [3H]UTP into protein and RNA, respectively, was increased 2-3-fold in isolated mitochondria from thiamphenicol-treated animals, indicating increased rates of synthesis of both. Electrophoretic analysis of transcripts labelled with [32P]UTP suggests that synthesis of all the transcripts is increased. The steady-state concentrations of mitochondrial transcripts, measured by Northern blotting using nick-translated cloned EcoRI fragments of rat liver mtDNA, were also elevated 2-4-fold in thiamphenicol-treated animals. The data suggest that mitochondrial transcription is under control of a mitochondrial factor which, in turn, is dependent upon mitochondrial protein synthesis.

Uncoupling effect of fungal hydroxyanthraquinones on mitochondrial oxidative phosphorylation.

Structure-uncoupling activity relationship of seven anthraquinone derivatives were investigated using rat liver mitochondria. Three compounds bearing the free hydroxyl group at the beta-position of their anthraquinone nucleus (1,3,6,8-tetrahydroxyanthraquinone, 1-acetyl-2,4,5,7-tetrahydroxy-9,10-anthracenedione and skyrin) exhibited uncoupling effect. Rugulosin, rugulin and physcion (all lacking the hydroxyl at the beta-position) were ineffective. Erythroglaucin, a derivative of physcion with the free hydroxyl group at the gamma-position, exhibited the highest uncoupling activity in the series tested. In addition, erythroglaucin abolished the energy dependent Ca2+ retention in mitochondria and induced Ca2+ leak. It also prevented the energization of mitochondrial membrane by ATP and induced a loss of the ATP induced membrane potential similarly as did carbonylcyanamide-3-chlorophenyl hydrazone (CCCP). The data show that the free hydroxyl group at either the gamma-position or the beta-position of anthraquinone nucleus is a prerequisite of the uncoupling activity of hydroxyanthraquinones.

The effect of inhibition of mitochondrial protein synthesis on the proliferation and phenotypic properties of a rat leukemia in different stages of in-vivo tumor development.

It has been shown before that prolonged treatment with doxycycline (DC), an inhibitor of mitochondrial protein synthesis, leads to proliferation arrest of a leukemia in the rat and, moreover, to eradication of this tumor. It has also been demonstrated that the period of treatment required to achieve this is shorter when DC administration is started in later stages of tumor progression. Therefore, the leukemic cells may have properties with regard to DC sensitivity which change with time during tumor progression. In the present study this hypothesis was tested by studying the permeability for DC, the presence of cell-surface molecules, and the mitochondrial content of the leukemic cells in various stages of tumor development in control and in DC-treated rats. Changes in DC permeability or antigenic phenotype were not observed, but the content of mitochondria decreases during tumor progression. DC treatment leads to an additional reduction of the content of functional mitochondria which results in proliferation arrest. The higher mitochondrial content of the leukemic cells during the earlier stages of tumor development explains thus why a longer period of DC treatment is needed to achieve growth arrest when treatment is started in these stages.

Rhodamine 6G inhibits the matrix-catalyzed processing of precursors of rat-liver mitochondrial proteins.

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the alpha and beta subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B.D. (1984) Eur. J. Biochem. 144, 387-392].

Elevated phosphate transporting activity and phosphate carrier content in mitochondria of rat hepatoma with high glycolytic capacity.

Transport of inorganic phosphate into Zajdela hepatoma mitochondria proceeds with approximately the same Km and about two times higher Vmax than the transport into mitochondria of rat liver. As detected by (a) titration of the inhibition of mitochondrial phosphate-stimulated respiration and phosphate-induced swelling by mersalyl and (b) binding of /14C/-NEM and /14C/-DCCD to a 33 kDa protein in mitochondria, the higher phosphate transporting activity of the hepatoma mitochondria is due to about a three fold increase in phosphate carrier content in the tumor mitochondria.

Interaction of cis-diamminedichloroplatinum (II) with mitochondrial phosphate carrier.

Effect of cis-Pt(II) on mitochondrial phosphate transport has been studied. The inhibition of transport by cis-Pt(II) is demonstrated by the effect of the drug on mitochondrial phosphate-induced swelling and respiration. A diminished 14C-NEM binding to 33 kD mitochondrial protein in the presence of cis-Pt(II) suggests that cis-Pt(II) inhibits mitochondrial phosphate transport by a direct interaction with mitochondrial phosphate carrier.

In vitro transport of F1-ATPase beta-subunit into mitochondria of Zajdela hepatoma and rat liver.

Transport of precursor of F1-ATPase beta-subunit into isolated mitochondria of Zajdela hepatoma and rat liver was examined. The hepatoma mitochondria were more active in the process than the liver organelles indicating that the relative F1-ATPase deficiency in the tumor mitochondria does not result from an impaired transport of F1-ATPase subunits into the tumor organelles. Similar results were obtained using digitonin-treated rat hepatocytes and Zajdela hepatoma cells instead of isolated mitochondria. The suitability of the digitonin-treated cells in the study of protein transport into mitochondria in vitro is demonstrated and the advantages of this system over isolated mitochondria are discussed.

Effect of cis-diamminedichloroplatinum (II), cyclophosphamide and tumor age on the rate of mitochondrial and overall cellular protein synthesis in Zajdela hepatoma.

The effect of in vivo treatment with cis-Pt(II) and/or cyclophosphamide on overall cellular and mitochondrial protein synthesis in Zajdela hepatoma was examined. The rate of the overall cellular protein synthesis decreased by about 60-80%, whereas the rate of the process in mitochondria was affected only marginally upon the treatment. Qualitatively similar changes in the relative rates of the two processes were found in Zajdela hepatoma cells during the ageing of the tumor.

Increased content of natural ATPase inhibitor in tumor mitochondria.

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria.

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.

Unequal inhibition of protein synthesis in mitochondria of Zajdela hepatoma and rat liver after in vivo treatment with cis-diamminedichloroplatinum (II).

The effect of in vivo administered cis Pt(II) on mitochondrial and overall cellular protein synthesis in hepatocytes from regenerating rat liver and Zajdela hepatoma was examined. In both types of cells the overall cellular protein synthesis was inhibited by the drug approximately to the same extent (about 50%). Protein synthesis in mitochondria of Zajdela hepatoma was practically unaffected by the drug, whereas that in rat liver was inhibited similarly as was the overall cellular protein synthesis. Cis Pt(II) treatment had no detectable effect on the electrophoretic pattern of peptides synthesized in mitochondria of rat liver and Zajdela hepatoma. Relative content of cytochrome oxidase subunit IV to subunit II in Zajdela hepatoma mitochondria decreased upon cis Pt(II) treatment. The amount of platinum bound to Zajdela hepatoma mitochondria upon in vivo cis Pt(II) treatment was about two times lower than that bound to mitochondria of rat liver. It is concluded that protein synthesis in Zajdela hepatoma mitochondria is more resistant to in vivo cis Pt(II) treatment than the process in mitochondria of rat liver, and that this effect results from lower binding of cis Pt(II) and/or its derivatives to the tumor mitochondria.

Rat liver protoporphyrinogen IX oxidase: site of synthesis and factor influencing its activity.

Rat liver protoporphyrinogen IX oxidase is not formed in mitochondria in contrast to the claims made for the yeast enzyme (Poulson and Polglase, FEBS Lett. (1974) 40, 258). Inhibition of mitochondrial protein synthesis in regenerating rat livers by thiamphenicol led, instead, to a slight increase in protoporphyrinogen oxidase activity. Protoporphyrinogen IX oxidase was not induced in rat liver by triiodothyronine, an inducer of mitochondrial protein synthesis, or by AIA, an inducer of heme synthesis. Significant increases in activity were observed to be associated with rapidly growing cells, such as regenerating livers and rat ascites hepatoma cells.

Effect of combined chemotherapy tested in partially hepatectomized rats bearing Zajdela hepatoma.

Ascitic Zajdela hepatoma growing in partially hepatectomized rats was used for testing cytostatics in single and two-drugs combination chemotherapy. At the optimal dosage the highest selective activity against tumor cells (hepatoma) with low inhibition of normal cells (regenerating liver cells) was seen in the combination cis-platinum + methotrexate. Synergistic effect of this combination was found when suboptimal dose of MTX was combined with low doses of cis-Pt. Dose-dependent DNA synthesis inhibition following i.p. administration of cis-Pt was documented by 3H-thymidine incorporation. Although the content of platinum expressed per DNA amount was four times higher in regenerating hepatocytes when compared with hepatoma cells, the growth inhibiting effect of cis-Pt was selectively expressed against the hepatoma cells.