Basis for decreased D-beta-hydroxybutyrate dehydrogenase activity in liver mitochondria from diabetic rats

Liver mitochondria from rats made diabetic with streptozotocin have a reduced level of D-beta-hydroxybutyrate dehydrogenase (BDH) activity and decreased ratios of oleic/stearic and arachidonic/linoleic acids in the phospholipids of the mitochondrial membrane. This altered activity and lipid environment result from insulin deprivation since maintenance of the diabetic rats on insulin leads to normal characteristics (J.C. Vidal, J.O. McIntyre, P.F. Churchill, and S. Fleischer (1983) Arch. Biochem, Biophys. 224, 643-658). In the present study, the basis for the reduced enzymatic activity of this lipid-requiring enzyme was analyzed using three approaches: (i) Purified D-beta-hydroxybutyrate, dehydrogenase was inserted into membranes from mitochondria, submitochondrial vesicles, and mitochondrial lipids extracted therefrom. The activation was the same and optimal irrespective of whether the preparations were derived from normal or diabetic rat liver. Therefore, the decreased activity does not appear to be referable to an altered lipid composition. (ii) BDH activity can be released from the mitochondria by phospholipase A2 digestion. The released activity was proportional to the endogenous activity in the submitochondrial vesicles from normal and diabetic membranes. (iii) The BDH activity in submitochondrial vesicles was titrated by inhibition with specific antiserum. Less enzyme was found in mitochondria from diabetic rats as compared with those from normal animals. Hence, the lowered enzymatic activity is due to decreased enzyme in the mitochondrial inner membrane and not to the modified lipid environment.     

Three proton pumps,morphology and movements

The diameter of F₁ coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F₁, then reconstituted by addition of F₁. The extramembranous size of F₁ is 9.7 nm and F₁ protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 µg per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.Abbreviations SMP submitochondrial vesicles prepared from bovine heart mitochondria exposed to sonic oscillation in the presence of pyrophosphate - F₁ the water-soluble coupling factor of the mitochondrial ATPase complex - CF₁ the water-soluble coupling factor of the chloroplast ATPase complex - ASU vesicles submitochondrial vesicles prepared from bovine heart mitochondria disrupted by sonic oscillation in ammonia, then passed through Sephadex and treated with urea - OSCP oligomycin sensitivity-conferring protein - Mega 8, 9, and 10 for octoylnanoyl, and decanoyl-N-methylglucamide - 1799 bis-(hexafluoroacetonyl)acetone - PMS N-methylphenazonium methosulfate     

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

The orientation of d-β-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane

d-β-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and ‘inside-out’ submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A₂ digestion than from mitochondria. Conclusive evidence that d-β-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome $\text{c}$. The arrangement of d-β-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that d-β-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.     

Plasma membranes from candida tropicalis grown on glucose or hexadecane. I. Isolation,identification and purification

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes.After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation.Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences.The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg²⁺-dependant ATPase was determined.By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues

Irrespective of the starting material, i.e. washed mitochondria, purified mitochondria or mitoplast from Solanum tuberosum L., submitochondrial particles of well-defined polarities can be generated by French press treatment in low-salt medium or by sonication in high-salt medium. The first treatment will result in submitochondrial particles which are more than 80% right-side-out (right-side-out particles), the second in submitochondrial particles that are more than 80% inside-out (inside-out particles). The isoelectric point (pI = 4.0) of the inside-out particles measured by cross-partition is distinctly different from the isoelectric points of the other mitochondrial membranes which exhibit pI values between 4.5 and 4.7. The surface charge density measured by 9-aminoacridine fluorescence varies in the same order from −27 mC · m⁻² for Percoll-purified mitochondria to −51 mC · m⁻² for both right-side-out and inside-out particles. Even though the charge densities for the two surfaces of the inner membrane are similar, inside-out particles are much more negatively charged at pH 7.0, since they are 6-times larger. These results clearly demonstrate that it is possible to obtain submitochondrial particles of various polarities and sizes which in turn constitute valuable tools for the study of lateral and transverse asymmetry of the inner mitochondrial membrane.     

Effects of lipids on the activity of soluble mitochondrial pyrophosphatase and its conversion to the membrane-bound form

The effects of lipids on the activity of soluble and membrane-bound pyrophosphatase from beef heart mitochondria were studied. An addition of total mitochondrial lipid, phosphatidyl choline, phosphatidyl ethanolamine or cardiolipin resulted in stimulation of the enzymatic activity and an increase in thermal stability of the soluble enzyme. The maximal activating effect was exerted by the total mitochondrial lipid and phosphatidyl choline. The electrophoretic data suggest that phosphatidyl choline is a component of membrane pyrophosphatase. Preincubation of the soluble enzyme with phosphatidyl choline converted the enzyme into a membrane form, which is capable to carry out the energy-dependent synthesis of PPi in submitochondrial particles.     

Lipid-protein interactions in mitochondria. The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis.

Phospholipase A₂ (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.     

Calorimetric studies of lipid phase transitions in native and heat-denatured membranes of beef heart submitochondrial particles

The lipids in beef heart submitochondrial particles undergo a broad reversible endothermic phase change centered at about −10°C. Following protein denaturation, a new reversible transition centered at about 20°C appears. The extracted lipids from these membranes exhibit thermal behavior that is essentially identical to the lipid transition in the intact membrane after protein denaturation. A role for this latent pool of higher-melting lipids is proposed.     

Structural studies on the organization of proteins in mitochondrial membranes using proteolytic enzymes

Proteolytic enzymes, pronase and trypsin, digest protein in ETP and in SU-particles (devoid of the soluble ATPase) at similar rates and to the same extents for intact and lipid-depleted membranes, showing that lipids do not constitute a barrier to the action of the proteases. The rates and extents of hydrolysis are slightly depressed when membranes are reconstituted from lipid-depleted particles and phospholipids. The hydrolysis rates for the various particles are not greatly enhanced by detergent solubilization nor by other denaturing treatments, indicating that the rates measured in absence of treatments are maximal under the conditions used. The circular dichroism spectra of pronase treated ETP are noticeably altered showing modification of the original conformation. Moreover, enzymic activities of mitochondria and submitochondrial particles are progressively affected by proteases according to their localization at, or near to, a given surface of the membrane. The matrix enzyme, malate dehydrogenase, is not apparently released from mitochondria during the initial incubation period. The results are tentatively discussed in terms of organization of lipids and proteins in the mitochondrial membrane.     

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.     

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids.

2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did not inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition.

3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols.

4. It is concluded that mitochondrial transhydrogenase, catalyzing the non-energy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation

A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles.     

Beef-heart submitochondrial particles: a mixture of mitochondrial inner and outer membranes.

1. EPR spectra at 9 GHz and 83 degrees K of NADH-reduced anaerobic beef-heart submitochondrial particles, prepared from mitochondria by sonication and centrifugation, contain a signal (gz equals to 2.01, gy equals to 1.94, gx equals to 1.89) due to an iron-sulphur center of the mitochondrial outer membrane. 2. The ratio of inner and outer membranes in submitochondrial particles is not greatly different from that in beef-heart mitochondria. 3. Beef-heart submitochondrial particles free from outer-membrane contamination have been prepared by free-flow electrophoresis. EPR spectra at 83 degrees K of such particles are presented.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Intramitochondrial phospholipid trafficking

Mitochondrial functions and architecture rely on a defined lipid composition of their outer and inner membranes, which are characterized by a high content of non-bilayer phospholipids such as cardiolipin (CL) and phosphatidylethanolamine (PE). Mitochondrial membrane lipids are synthesized in the endoplasmic reticulum (ER) or within mitochondria from ER-derived precursor lipids, are asymmetrically distributed within mitochondria and can relocate in response to cellular stress. Maintenance of lipid homeostasis thus requires multiple lipid transport processes to be orchestrated within mitochondria. Recent findings identified members of the Ups/PRELI family as specific lipid transfer proteins in mitochondria that shuttle phospholipids between mitochondrial membranes. They cooperate with membrane organizing proteins that preserve the spatial organization of mitochondrial membranes and the formation of membrane contact sites, unravelling an intimate crosstalk of membrane lipid transport and homeostasis with the structural organization of mitochondria.This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.