Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.     

The links between membrane composition, metabolic rate and lifespan.

The acyl composition of tissue phospholipids varies in a systematic manner among species. Phospholipids, and thus membrane bilayers, from the tissues of small mammal and bird species have a high content of docosahexaenoic acid (DHA) compared to large species. A similar difference exists between the tissues of endothermic mammals and ectothermic reptiles. High DHA content in phospholipids is associated with high metabolic activity and this observation has led to the development of the "membrane pacemaker" theory of metabolism. This proposes that highly polyunsaturated acyl chains impart physical properties to membrane bilayers that enhance and speed up the molecular activity of membrane proteins and consequently the metabolic activity of cells, tissues and the whole animal. The brain has highly polyunsaturated membranes irrespective of body size and possible reasons for this are discussed. Highly polyunsaturated acyl chains are very susceptible to peroxidative damage. It is suggested that these chemical properties of highly polyunsaturated membrane acyl chains have important implications for understanding aging and the determination of longevity.     

The contribution of the leak of protons across the mitochondrial inner membrane to standard metabolic rate

This paper presents and assesses the hypothesis that the proton leak across the mitochondrial inner membrane is an important contributor to standard metabolic rate, and that increases in the amount of mitochondrial inner membrane may be important in causing changes in proton leak and in the standard metabolic rate. The standard metabolic rate of an animal is known to be a function of body mass, phylogeny and thyroid status, and is largely attributed to the metabolically active internal organs. The total area of mitochondrial inner membrane in these organs correlates well with standard metabolic rate over a wide range of body masses in both ectotherms and endotherms. In hepatocytes isolated from rats, proton leak across the mitochondrial inner membrane accounts for about 30% of the resting oxygen consumption, and the distribution of control over respiration suggests that changes in mitochondrial inner membrane surface area will be accompanied by significant changes in the proton leak. This change in the leak will result in significant changes in resting oxygen consumption, but changes in ATP demand may also have a role to play in determining resting respiration rate. Extrapolation of these results to other tissues and other animals suggests that the hypothesis has the potential to explain a substantial proportion of the variation in standard metabolic rate with body mass, phylogeny and thyroid status. However, in most cases the quantitative contribution of proton leak compared to cellular ATP turnover has yet to be experimentally determined.     

The export of metabolites from mitochondria and anaplerosis in insulin secretion

Combinations of insulin secretagogue-derived metabolites were added to microgram amounts of mitochondria obtained from rat and mouse pancreatic islets and the INS-1 cell line, and the export of citric acid cycle intermediates was surveyed to study anaplerosis in insulin secretion. Cellular levels of metabolites were also measured. In mitochondria from all three tissues, malate production was the most responsive to various substrates. The export of citrate and isocitrate in the presence of pyruvate and most other substrates was small and their levels in intact cells did not change with any secretagogue, except in INS-1 cells where citrate increased slightly. Changes in alpha-ketoglutarate and glutamate export from mitochondria and levels in intact cells indicate that glutamate can be consumed as a fuel secretagogue, but it is not likely produced as a messenger in insulin secretion. The citrate level may not need to increase in order to provide increased malonyl-CoA for signaling insulin secretion. Unlike some cells, insulin cells probably obtain cytosolic NADPH equivalents by exporting them from mitochondria to the cytosol via a pyruvate malate shuttle or an isocitrate shuttle. Only fuels that can enhance anaplerosis via pyruvate or alpha-ketoglutarate can be insulin secretagogues.     

Synthesis of phospholipids in mitochondria and other membrane fractions of rabbit reticulocytes.

1. Reticulocytosis of 40-50% was obtained in rabbits by daily bleeding. Reticulocytes (plus erythrocytes) were subfractionated into plasma membrane fraction, mitochondria and the post-mitochondrial fraction. 2. In all fractions, fatty acids were incorporated into phospholipids. This process was ATP dependent and represented acylation of lysophospholipids. 3. Incorporation of fatty acids into lysophosphatidic and phosphatidic acids occurred only in the presence of sn-glycerol 3-phosphate and was observed in mitochondria and the post-mitochondrial fraction. It represents a two-step acylation of sn-glycerol 3-phosphate. 4. Incorporation of phosphorylcholine from CDPcholine into phosphatidylcholine was observed in the mitochondrial and the post-mitochondrial fractions. This activity was correlated with NADPH-cytochrome c reductase and was probably connected with the remnants of the endoplasmic reticulum.     

Cytochemical studies of mitochondria. I. The separation and identification of a membrane fraction from isolated mitochondria

Mitochondria isolated from rat liver and suspended in 0.44 M sucrose were disrupted by treatment with 0.3 per cent Na deoxycholate. The treated suspension was fractionated by differential centrifugation into a number of fractions and the respective pellets were examined in sections in the electron microscope. One of these fractions was found to consist of apparently membrane-bound (vesicular) elements. The difference between interfaces and membranes was discussed and the material of this fraction was found to meet stated requirements identifying it as membranous. A detailed study of the disruption process undergone by mitochondria in the presence of Na deoxycholate showed that the elements of this fraction were derived from structural elements assumed to be mitochondrial membranes. The findings thus demonstrate that mitochondria do possess membranes as defined and that these membranes can be isolated in a relatively pure form.     

The cationically selective state of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin.

The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.     

Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria.

The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C.     

Continuous pyruvate carbon flux to newly synthesized cholesterol and the suppressed evolution of pyruvate-generated CO2 in tumors: further evidence for a persistent truncated Krebs cycle in hepatomas

Viable tissue slices from rat liver and Morris hepatoma 3924A were compared as to their ability to incorporate carbons from [U-14 C]pyruvate into newly synthesized cholesterol versus CO2. By 4 h, the tumor slice incubation had incorporated over 6-fold more pyruvate carbons into the sterol than into CO2, relative to the normal liver slice incubation, per g tissue protein. However, the presence of the mitochondrial citrate exchange carrier inhibitor 1,2,3-benzenetricarboxylate in the incubation inhibited the formation of [14C]cholesterol, while simultaneously leading to an increase in the rate of 14CO2 production in the tumor. In the normal liver system by contrast, benzenetricarboxylate also inhibited [14C]cholesterol formation, but had hardly any effect on the already high rate of 14CO2 production. The ability of benzenetricarboxylate to inhibit the rapid carbon flux from pyruvate to cholesterol, and to steer the metabolic flow of carbons toward oxidative decarboxylation via the Krebs cycle in whole, viable tumor tissue, indirectly emphasizes the importance of the mitochondrial citrate exchange carrier in supporting the decontrol of cholesterogenesis de novo in tumors by accelerating the supply of lipogenic precursor carbons to the tumor cytosol. These studies may be therefore interpreted as extensions, to the level of whole-cell metabolism, of the concept of a persistent 'truncated' Krebs cycle in the mitochondria of metastatic cancer tissue. This concept states, in part, that a rapid efflux of mitochondrially generated citrate would operate preferentially in tumors, and thus provide carbons continuously to the cytoplasmic compartment where the well-established deregulated pathway of cholesterogenesis occurs (Parlo, R.A. and Coleman, P.S. (1984) J. Biol. Chem. 259, 9997-10003; Coleman, P.S. and Lavietes, B.B. (1981) CRC Crit. Rev. Biochem. 11, 341-393).     

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

A mathematical model of the pyruvate oxidation in liver mitochondria. 1. Regulation of the Krebs cycle by adenine and pyridine nucleotides]

A mathematical model is proposed to describe the behavior of the pyruvate metabolic reactions, Krebs cycle and oxidative phosphorylation over a wide range of changes in the pyruvate influx rate and the activities of ATPase and NADH-reoxidating dehydrogenase. The role of adenine and pyridine nucleotides in various allosteric regulations of the Krebs cycle enzymes is discussed. The accumulation of ATP and NADH has been shown to proceed in definite succession, which makes the allosteric regulation of the Krebs cycle enzymes successive too. First "works" the inhibition by ATP, then by NADH. It has been shown that the properties of the model are in qualitative agreement with the experimental data (Garber A., Hanson R. [1]) on pyruvate oxidation by mitochondria from guinea pig liver, when allosteric regulation of isocitrate dehydrogenase by adenine nucleotides is taken into account.     

Effect of the oxidation of glycine and Krebs cycle substrates on cytochrome activity and alternative pathways of mitochondria from leaves of Pisum sativum L

Isolated mitochondria from adult leaves of Pisum sativum had the capacity to oxidize simultaneously glycine and several substrates of the Krebs cycle (e.g. malate, succinate, citrate, 2-oxo-glutarate), either in the presence of ADP (state three) or in the absence of ADP (state four). The sensitivity of the mitochondrial respiration to inhibitors of the cytochrome (e.g. antimycin A) and the alternative (e.g. salicylhydroxamic acid, SHAM, and tetraethylthiuram disulfide, disulfiram) pathways varied depending on the substrate(s) being used. For instance, the rate of oxygen uptake resistant to antimycin A, which is an estimate of the capacity of the alternative pathway, varied depending on whether glycine was added or not to a medium with malate and succinate. The state four rate of oxygen consumption in the presence of malate or succinate was greatly stimulated by the addition of glycine, and vice versa. This stimulation was apparently mediated by the alternative pathway. The results suggest that part of the electron transport capacity (including the alternative pathway) of these mitochondria is specifically associated with glycine oxidation, and therefore with photo-respiration.     

The effect of starvation and alloxan-diabetes on the contents of citrate and other metabolic intermediates in rat liver

1. The content of citrate in ;freeze-clamped' livers from starved and alloxan-diabetic rats was measured by using the specific citrate assay method of Gruber & Moellering (1966). 2. The content of citrate fell progressively during a period of 48hr. starvation to reach a plateau value that is 50% of the value for livers from fed rats. Some possible explanations for the conflicting reports of changes in hepatic citrate content during starvation are discussed. 3. The hepatic contents of ATP, pyruvate, lactate, glycogen and the hexose phosphates were decreased during starvation, whereas those of acetyl-CoA and AMP were increased. 4. Acute alloxan-diabetes produced similar changes in the contents of these metabolic intermediates. 5. The effects of starvation and diabetes on the citrate and acetyl-CoA contents are discussed in relation to control of gluconeogenesis, fatty acid synthesis and the activity of citrate synthase.     

The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation.

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.