Štefan Kužela

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 the 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.

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.

Some properties of coupled hepatoma mitochondria exhibiting uncoupler-insensitive ATPase activity

Summary Coupled mitochondria of Zajdela hepatoma exhibited ATPase activity which was not stimulated by uncouplers. In spite of this fact the properties of respiration, energy-dependent Ca2+ uptake and adenine nucleotide translocation as well as electrophoretic pattern of membrane proteins of Zajdela hepatoma mitochondria did not differ from those of Ehrlich ascites tumour mitochondria which possess uncoupler-sensitive ATPase.

Amino acid incorporation by isolated rat liver mitochondria into two protein components of mitochondrial ATPase complex: Lack of incorporation into dicyclohexylcarbodiimide binding proteolipid

The role of products of mitochondrial protein synthesis in the biogenesis of the mitochondrial ATPase complex of yeast and fungi is well documented. In Saccharomyces cerevisiae four [ 1 ] and in Neurospora crassa two [2] protein subunits of the complex are synthesized intramitochondrially. Special attention has been paid to the elucidation of the site of the synthesis of DCCD binding proteolipid of the mitochondrial ATPase complex. In yeast [3] this component is synthesized within mitochondria and in fungi [4,5] outside the organelles. Less information exists on the participation of mitochondrial translation products in the formation of mitochondrial ATPase complex and on the site of the synthesis of DCCD binding proteolipid of the complex in animal cells. The intramitochondrial formation of three protein components of mitochondrial ATPase complex in oocytes [6] and in regenerating rat liver [7] has been indicated by selective invivo inhibition of cytoplasmic or mitochondrial protein synthesis. Incorporation of labeled amino acids by isolated rat [8] and mouse [9] liver mitochondria into DCCD binding proteolipid has been described and it was concluded that the DCCD binding component of rat [8] and mouse [9] liver mitochondrial ATPase complex represents a product of mitochondrial protein synthesis. On the other hand, selective in vivo inhibition of mitochondrial protein synthesis in regenerating rat liver did not affect significantly amino

Low translational efficiency of the F1‐ATPase β‐subunit mRNA largely accounts for the decreased ATPase content in brown adipose tissue mitochondria

The half‐life of the F1‐ATPase β‐subunit (F1‐β) mRNA in ATPase‐poor brown adipose tissue (BAT) (t?? = 9.5 h) was found to be 3–7‐fold shorter than in liver (t?? = 27 h) and heart (t?? = 63 h) of mice. When translated in reticulocyte lysate, a 2–3‐fold lower efficiency appeared with F1‐β mRNA from BAT than from other tissues. The in vitro synthesized F1‐β 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 or abundant F1‐β mRNA in BAT is not fully translatable most likely due to its low metabolic stability.

Differentiation of dicyclohexylcarbodiimide reactive sites of the ATPase complex in bovine heart mitochondria

1. In isolated bovine heart mitochondria, the 14C-labelled dicyclohexylcarbodiimide (DCCD) induced inhibition of the ATPase activity is accompanied by labelling of three polypeptides of Mx 9000, 16 000 and 33 000. Of these, only the 9000 polypeptide reacts with [14C]DCCD proportionally to the inhibitory effect, being saturated when the enzyme is maximally inhibited. 2. The 9000 and 16 000 polypeptides are extracted by neutral chloroform/methanol (2 : 1 v/v) while the 33 000 polypeptide remains in the non-extractable residue. No disaggregation of the polypeptides takes place during the extraction. 3. In the ATPase complex immunoprecipitated with antibody against F1, the 9000 and 16 000 polypeptides are present, but the 33 000 polypeptide is absent. 4. The results obtained indicate that the 33 000 polypeptide is not a component of the ATPase complex. As far as F0 is concerned, two types of the binding sites for DCCD were demonstrated, corresponding to the 9000 and 16 000 polypeptides. Their existence is explained by a non-random arrangement among individual monomers of the DCCD-binding protein.

Solubility of the intramitochondrially synthesized protein and other membrane proteins of rat liver mitochondria in acidic or neutral chloroform-methanol.

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, v v /, 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 in vivo is presented.

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.

The binding of dicyclohexylcarbodiimide to uncoupling protein in brown adipose tissue mitochondria

The generation of heat in brown adipose tissue has been related to the uncoupling of respiration in their mitochondria (reviews [ 1,2]). The uncoupling in these mitochondria is achieved by a high ion conductance of the mitochondrial membrane and is specifically controlled by exogenous purine nucleotides [3-71. The binding site for these nucleotides was identified as a 32 000 Mr protein that is present in considerable amount only in brown adipose tissue mitochondria [S111. It is proposed that this protein (so called GDP-binding or uncoupling protein) is directly responsible for the physiological uncoupling of brown adipose tissue mitochondria. Here, we report that N,N’-dicyclohexylcarbodiimide a well-established inhibitor or proton-pumping activity of several membrane enzymes [12-161, binds rather specifically to the uncoupling protein in brown adipose tissue mitochondria. In addition, it inhibits the high chloride permeability of brown adipose tissue mitochondrial membrane. We propose that the 32 000 Mr uncoupling protein may function in mitochondrial membrane of brown adipose tissue similarly to other proton-translocating DCCD binding proteins.

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.