ABSTRACT Almost all protein species of submitochondrial particles from rat liver identified by SD... more ABSTRACT Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, /, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver is presented.
Rat liver protoporphyrinogen IX oxidase is not formed in mitochondria in contrast to the claims m... more 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.
The effect of the yeast cell-death inducing agents, Bax and acetic acid, on mitochondrial structu... more The effect of the yeast cell-death inducing agents, Bax and acetic acid, on mitochondrial structure of Schizosaccharomyces pombe was studied. Comparison of mitochondrial structures in cells grown on different substrates and visualized with different probes revealed variations in their morphology. Cells grown on respiratory C sources as well as in the presence of antimycin A exhibited punctuated mitochondria when visualized with mitochondrially targeted green fluorescent protein, while they still appeared as tubular structures when stained with DiOC6(3). Both expression of Bax and acetic acid treatment induced fragmentation and aggregation of mitochondrial network, which could be prevented by coexpression of Bcl-XL. Aberrant mitochondrial morphology generated by either Bax or acetic acid was not accompanied with the loss of mitochondrial genome (mtDNA), indicating that alterations of mitochondrial morphology following death stimuli follow different mechanisms than those involved in mitochondrial inheritance mutants.
The administration of chloramphenicol in high doses (300 mg per kg body weight in 6 hour interval... more The administration of chloramphenicol in high doses (300 mg per kg body weight in 6 hour intervals) to partially (70%) hepatectomized rats resulted in a marked stimulation of liver regeneration.
Hydrolysis of extramitochondrial ATP by coupled Zajdela hepatoma mitochondria is not stimulated b... more Hydrolysis of extramitochondrial ATP by coupled Zajdela hepatoma mitochondria is not stimulated by uncouplers of oxidative phosphorylation. The results of the present study show that the hydrolysis of intramitochondrial ATP in these mitochondria is stimulated by DNP and CCCP. It is proposed that the uncoupler insensitivity of ATPase in coupled Zajdela hepatoma mitochondria with exogenous ATP as a substrate result from an altered functional relationship between ATPase and ADP, ATP translocase.
The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma... more The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma were compared. The specific activity of ATPase in sonicated mitochondria and in mitochondrial membrane fraction of rat liver was almost two times higher than the specific activity in the corresponding fraction of Zajdela hepatoma. Accordingly, the autovertin binding capacity of rat liver mitochondrial membrane fraction as well as the yield of F1-ATPase from this fraction were about two times higher than those of the mitochondrial membrane fraction of Zajdela hepatoma. The results show that mitochondria of Zajdela Hapatoma possess about half amount of ATPase present in rat liver mitochondria.
The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effe... more The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-x(L), when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-x(L) is...
ABSTRACT SUMMARY 1. Close similarities between yeast and mammalian mitochondria were found with r... more ABSTRACT SUMMARY 1. Close similarities between yeast and mammalian mitochondria were found with respect to (a) osmotic response i n impermeable solutes, sorbitol and KC1, (b) substrate translocation, (c) properties of the adenine nucleotide translocation system. A separate transport system for succinate, distinct from the dicarboxylate translocator, may be present i n yeast mitochondria. 2. Substrate translocation was found to be preserved in pro-mitochondria from anaerobically-grown cells and i n mitochondria from a respiration-deficient mutant. Adenine nucleotide translocation was demonstrated not t o be affected by the cytoplasmic mutation. Along w i t h previous data of other investigators, these results allow a general conclusion that neither the presence of a functional respirat o r y chain nor mitochondrial protein synthesis are prerequisite for the proper assemblage of the translocation systems in the mitochondrial membrane and for determining its permeability characteristics.
A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma... more A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma membranes Saccharomyces cerevisiae and during centrifugation of the solubilized membrane extract through a sucrose gradient, is separated from the Mr = 100,000 ATPase catalytic polypeptide by subsequent DEAE-cellulose chromatography. The purified casein kinase activity exhibits a low Km of 12 microM MgATP, is maximally stimulated by 6 mM free Mg2+, and is 50% inhibited by 300 microM Zn2+, by 7.5 micrograms of heparin/ml, and by 300 microM orthovanadate. It phosphorylates only seryl residues. The purified casein kinase contains two polypeptides of Mr = 45,000 and 39,000 which yield antibodies which do not cross-react to each other. The two polypeptides seem to originate from a precursor of Mr = 85,000 which is detected by both antibodies in partly purified fractions. In the absence of casein, a zinc and heparin-sensitive phosphorylation of the ATPase polypeptide is observed in partly pur...
The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocato... more The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1...
ABSTRACT Almost all protein species of submitochondrial particles from rat liver identified by SD... more ABSTRACT Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, /, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver is presented.
Rat liver protoporphyrinogen IX oxidase is not formed in mitochondria in contrast to the claims m... more 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.
The effect of the yeast cell-death inducing agents, Bax and acetic acid, on mitochondrial structu... more The effect of the yeast cell-death inducing agents, Bax and acetic acid, on mitochondrial structure of Schizosaccharomyces pombe was studied. Comparison of mitochondrial structures in cells grown on different substrates and visualized with different probes revealed variations in their morphology. Cells grown on respiratory C sources as well as in the presence of antimycin A exhibited punctuated mitochondria when visualized with mitochondrially targeted green fluorescent protein, while they still appeared as tubular structures when stained with DiOC6(3). Both expression of Bax and acetic acid treatment induced fragmentation and aggregation of mitochondrial network, which could be prevented by coexpression of Bcl-XL. Aberrant mitochondrial morphology generated by either Bax or acetic acid was not accompanied with the loss of mitochondrial genome (mtDNA), indicating that alterations of mitochondrial morphology following death stimuli follow different mechanisms than those involved in mitochondrial inheritance mutants.
The administration of chloramphenicol in high doses (300 mg per kg body weight in 6 hour interval... more The administration of chloramphenicol in high doses (300 mg per kg body weight in 6 hour intervals) to partially (70%) hepatectomized rats resulted in a marked stimulation of liver regeneration.
Hydrolysis of extramitochondrial ATP by coupled Zajdela hepatoma mitochondria is not stimulated b... more Hydrolysis of extramitochondrial ATP by coupled Zajdela hepatoma mitochondria is not stimulated by uncouplers of oxidative phosphorylation. The results of the present study show that the hydrolysis of intramitochondrial ATP in these mitochondria is stimulated by DNP and CCCP. It is proposed that the uncoupler insensitivity of ATPase in coupled Zajdela hepatoma mitochondria with exogenous ATP as a substrate result from an altered functional relationship between ATPase and ADP, ATP translocase.
The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma... more The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma were compared. The specific activity of ATPase in sonicated mitochondria and in mitochondrial membrane fraction of rat liver was almost two times higher than the specific activity in the corresponding fraction of Zajdela hepatoma. Accordingly, the autovertin binding capacity of rat liver mitochondrial membrane fraction as well as the yield of F1-ATPase from this fraction were about two times higher than those of the mitochondrial membrane fraction of Zajdela hepatoma. The results show that mitochondria of Zajdela Hapatoma possess about half amount of ATPase present in rat liver mitochondria.
The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effe... more The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-x(L), when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-x(L) is...
ABSTRACT SUMMARY 1. Close similarities between yeast and mammalian mitochondria were found with r... more ABSTRACT SUMMARY 1. Close similarities between yeast and mammalian mitochondria were found with respect to (a) osmotic response i n impermeable solutes, sorbitol and KC1, (b) substrate translocation, (c) properties of the adenine nucleotide translocation system. A separate transport system for succinate, distinct from the dicarboxylate translocator, may be present i n yeast mitochondria. 2. Substrate translocation was found to be preserved in pro-mitochondria from anaerobically-grown cells and i n mitochondria from a respiration-deficient mutant. Adenine nucleotide translocation was demonstrated not t o be affected by the cytoplasmic mutation. Along w i t h previous data of other investigators, these results allow a general conclusion that neither the presence of a functional respirat o r y chain nor mitochondrial protein synthesis are prerequisite for the proper assemblage of the translocation systems in the mitochondrial membrane and for determining its permeability characteristics.
A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma... more A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma membranes Saccharomyces cerevisiae and during centrifugation of the solubilized membrane extract through a sucrose gradient, is separated from the Mr = 100,000 ATPase catalytic polypeptide by subsequent DEAE-cellulose chromatography. The purified casein kinase activity exhibits a low Km of 12 microM MgATP, is maximally stimulated by 6 mM free Mg2+, and is 50% inhibited by 300 microM Zn2+, by 7.5 micrograms of heparin/ml, and by 300 microM orthovanadate. It phosphorylates only seryl residues. The purified casein kinase contains two polypeptides of Mr = 45,000 and 39,000 which yield antibodies which do not cross-react to each other. The two polypeptides seem to originate from a precursor of Mr = 85,000 which is detected by both antibodies in partly purified fractions. In the absence of casein, a zinc and heparin-sensitive phosphorylation of the ATPase polypeptide is observed in partly pur...
The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocato... more The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1...
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