Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization.

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the beta-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F1F0-ATPase and the lysosomal V-type ATPase.     

Identification and characteristics of a novel mitochondrial ATPase in rat liver

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.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 ± 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the β-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F₁F₀-ATPase and the lysosomal V-type ATPase.     

Adenosine triphosphatase from rat liver mitochondria - evidence for a mercurial-sensitive site for the activating anion bicarbonate.

The ATPase activity of purified mitochondrial ATPase (F₁) of rat liver is inhibited less than 15% by sulfhydryl reagents when assayed in TrisCl buffer. In Trisbicarbonate buffer the ATPase activity of the enzyme is two- to three-fold higher than in TrisCl. Significantly, the ATPase activity stimulated by bicarbonate can be inhibited by mercurial agents such as p-chloromercuribenzoate. The number of sulfhydryl groups accessible to ¹⁴C-p-chloromercuribenzoate is the same in TrisCl and Trisbicarbonate buffers. These experiments suggest that mercurials most likely inhibit bicarbonate-stimulated ATPase activity by blocking a site associated with bicarbonate binding rather than by blocking distinct sulfhydryl-sensitive hydrolytic sites induced by bicarbonate.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

The dicyclohexylcarbodiimide-binding protein of rat liver mitochondria as a product of the mitochondrial protein synthesis.

A product of mitochondrial protein synthesis in rat liver mitochondria, characterized by a low molecular weight (Mr is less than 10000) and an unusually high hydrophobicity, has been identified as the dicyclohexylcarbodiimide-binding protein and as a peptide of the hydrophobic sector of the mitochondrial ATPase complex. The purified protein still possesses the ability of bind dicyclohexylcarbodiimide.     

Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon.

The 7- to 10-fold increase in the rat liver serine:pyruvate aminotransferase activity after glucagon administration was shown to occur mainly in the mitochondrial matrix of parenchymal cells. The enzyme was purified from glucagon-treated rat liver mitochondria to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A specific rabbit antibody was prepared against the purified enzyme. Upon Ouchterlony double diffusion analysis, the mitochondrial extracts of glucagon-treated rat liver produced a single and fused precipitin line between the purified enzyme against the antibody. The supernatant fraction of glucagon-treated rat liver and the mitochondrial extracts of normal liver were also shown to make a single and fused precipitin line with the purified enzyme, when applied in large quantities. The quantitative immunotitration demonstrated that the glucagon-induced increase in the activity of liver serine:pyruvate aminotransferase were accompanied by the parallel increase in the amount of the enzyme antigen. Isotopic leucine incorporation studies showed that the relative rate of synthesis of the enzyme was increased approximately 10-fold by glucagon administration under the conditions employed. The rate of the degradation of the aminotransferase in the normal rat liver was a relatively slow process with a half-life of approximately 30 h. Thus the accumulation of serine:pyruvate aminotransferase in rat liver mitochondria by glucagon treatment can be ascribed mainly to the rise in the rate of enzyme synthesis.     

Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.     

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.     

Tetradifon: an oligomycin-like inhibitor of energy-linked activities of rat liver mitochondria

Tetradifon (p-chlorophenyl-2,4,5-trichlorophenyl sulfone) at concentrations between 4.5 and 27.0 nmoles/mg mitochondrial protein provides half-maximal inhibition of the following energy-linked activities of rat liver mitochondria: ADP-stimulated respiration, DNP-stimulated ATPase activity, Mg⁺⁺-stimulated ATPase activity, and P_i-ATP exchange activity. Tetradifon has no effect on the activity of soluble ATPase purified from rat liver mitochondria. Respiration inhibited by tetradifon is restored upon addition of 2,4-dinitrophenol. It is concluded that tetradifon acts at or near the oligomycin sensitivity conferring complex located in the mitochondrial inner membrane.     

Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver

1. A basic protein (pI = 9.0) exhibiting superoxide dismutase activity was purified to homogeneity from rat liver by DEAE-cellulose, CM-cellulose and S-hexylglutathione affinity gel chromatography, chromatofocusing and Sephadex G-150 gel filtration. 2. The purified enzyme had specific activity of 4700 units/mg protein. The activity was not affected by 2 mM KCN. Manganese was detected in the enzyme preparation; the content was 0.9 mol/mol subunit. The N-terminal sequence of the first 23 amino acids of the enzyme exhibited a strong homology (except at position 11) with the mature protein of human Mn-superoxide dimutase. It is, therefore, concluded that the purified enzyme is Mn-superoxide dismutase. 3. The N-terminal amino acid sequence showed that about 50% of tyrosine at position 11 was substituted by glutamine, suggesting the existence of microheterogeneity of the superoxide dismutase protein. 4. The superoxide dismutase purified here was found to consist of subunits with an apparent relative molecular mass of 25,000. This larger than the value hitherto reported for rat liver Mn-superoxide dismutase (Mr 2,400); the previous low value is attributed to differences in methods. 5. The enzyme was shown by immuno-blotting to be exclusively localized in the mitochondrial fraction in the liver. The tissue content of Mn-superoxide dismutase is organ-specific, and was the highest in heart. The precursor protein of the Mn-superoxide dismutase was not detectable in the liver cytosolic and mitochondrial fractions as well as in several extrahepatic organs (lung, heart, brain, muscle, kidney and testis), suggesting rapid transport across mitochondrial membranes and processing of the superoxide dismutase protein.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.     

Adenosine triphosphatase of rat liver mitochondria: detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme.

The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 mumol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37 degrees C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 mumol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201000 g for 90 min, and readily passes through a 250-A Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 muM oligomycin. In addition, inhibitions of 60% or better are obtained in the presence of 1-8 muM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100 this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive. ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycin-sensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria.     

Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium

This study reports on the distribution of bicarbonate-stimulated ATPase in rat intestinal epithelial cells. Brush-border membranes and basolateral membranes were separated from each other and from mitochondrial and other intracellular membranes by differential and density gradient centrifugation. Bicarbonate-sensitive ATPase activity followed the mitochondrial marker succinic dehydrogenase closely throughout all the centrifugation steps. The low HCO3--ATPase activity in purified brush-border and basolateral plasma membranes could be accounted for quantitatively by the small mitochondrial contamination. Consequently, there are no grounds for postulating that this enzyme has a direct role in H+ or HCO3- transport across the rat small intestine.     

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.