Studies on the relationship between the inner and outer membranes of rat liver mitochondria as determined by subfractionation with digitonin

The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.     

Membrane-associated proteins affect the formation of filipin-cholesterol complexes in viral membranes

The presence and distribution of cholesterol in biological membranes can be visualized by complex formation with the polyene antibiotic filipin after or during fixation with glutaraldehyde. In the envelopes of budding and immature retroviruses no filipin-cholesterol complexes are formed, but in the plasma membrane of host cells and in the envelopes of mature viruses filipin-cholesterol complexes are easily detected. However, after treatment of glutaraldehyde-fixed cells with pepsin, the presence of cholesterol in the envelopes of budding and immature viral particles could also be demonstrated. This indicates that in these structures the reaction of cholesterol with filipin is inhibited by proteins associated with the cholesterol-containing membrane. Treatment of fixed cells with trypsin, and of unfixed cells with cytochalasin B (CB) had no effect on detectability of cholesterol in these structures. On no occasion were cholesterol-filipin complexes formed in coated pits. The present findings call for caution when interpretating data on absence of filipincholesterol complexes in those membrane domains that are characterized by the presence of closely associated proteins.     

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.     

Pyruvate carboxylase in the yeast pyc mutant

Pyruvate carboxylase deficiency was previously reported to be the biochemical lesion in a yeast mutant, designated pyc, which cannot utilize ethanol, acetate, pyruvate, aspartate, or oxaloacetate as the sole carbon source [C. Wills and T. Melham (1985) Arch. Biochem. Biophys. 236, 782-791; C. Wills et al. (1986) Arch. Biochem. Biophys. 246, 306-320]. We present evidence here that the level of pyruvate carboxylase activity as well as the native and subunit molecular weights of this enzyme are identical in the mutant and the wild type. In addition we have used immunocytochemical labeling to demonstrate the exclusively cytosolic localization of this enzyme in both the mutant and wild-type yeast.     

Modified Method for Purifying Rat Liver Branched-Chain α-Ketoacid Dehydrogenase Complex

Branched-chain α-ketoacid dehydrogenase complex (BCKDC) is a rate-limiting enzyme in the branched-chain amino acid catabolic pathway. We have developed a method of BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophys., 283, 293–299 (1990)). Here we report a modification of the method designed to obtain the purified enzyme with high reproducibility.     

Characterization of a superoxide dismutase mimic prepared from desferrioxamine and MnO2

The physicochemical properties of a soluble green complex obtained from the reaction of MnO2 and desferrioxamine have been investigated. The superoxide dismutase mimetic activity of this complex has been previously reported [D. Darr et al. (1987) Arch. Biochem. Biophys. 258, 351-355]. Optical spectra, titration experiments, and metal analyses are consistent with the presence of one gram-atom Mn3+ per mole of desferrioxamine. At least one of the hydroxamate groups is oxidized in the green complex. Reaction of Desferal with MnO2 in the presence of ascorbate yields a more active, pink complex. This pink complex is more stable toward EDTA, suggesting that in it all three hydroxamate groups are intact and ligated to the manganese. The physicochemical properties of these complexes were examined and structures are suggested.     

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.     

The tight junction as a barrier to cholesterol in canine epithelial cells

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.     

Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: a study using the filipin-cholesterol complex

Using filipin and freeze-fracture electron microscopy, we examined the distribution of membrane cholesterol during the fusion of myoblasts in vitro. The early stages of fusion were characterized by the depletion of cholesterol from the membrane apposition sites, at which the plasma membranes of two adjacent cells were in close contact. At first, filipin-cholesterol complexes were absent from the plasma membrane of one cell only and were distributed homogeneously on the membrane of the other cell. Eventually, both of the closely apposed membranes became almost completely free the filipin-cholesterol complexes. Membrane fusion took place at several points within the filipin-cholesterol complex-free areas. In later stages, the cytoplasms of the fusing cells became confluent by fenestration of the plasma membranes formed with the filipin-cholesterol complex-free regions. Our observations suggest that membrane cholesterol is reorganized at these fusion sites and that fusion initiated by the juxtaposition of the cholesterol-free areas of each plasma membrane of the adjacent cells.     

Evidence against the proposed interaction of thionitrobenzoate with protein disulphide bonds

Thionitrobenzoate was prepared from 5,5'-dithiobis-(2-nitrobenzoic acid), and its reaction with several disulphide-containing proteins was examined under various conditions. No evidence for any cleavage of disulphide bonds was obtained. The claim by Robyt et al. [Arch. Biochem. Biophys. (1971) 147, 262-269] that thionitrobenzoate allows the quantitative determination of disulphide bonds in proteins is not substantiated.     

Factors affecting oxygen-induced changes in the activity of CTP-dependent lipid kinases in yeast

Previous work from this Laboratory (Szkopińska et al., 1988, Arch. Biochem. Biophys., 266, 124-131) indicated that CTP is a phosphate donor for the synthesis of phosphatidic acid and dolichyl phosphates. The elucidation of the role of mitochondrial membranes and mitochondrial proteins (isolation of rho- mutant) as well as specific detergents and sterols has been the aim of this work.     

Permeabilizing action of filipin III on model membranes through a filipin-phospholipid binding.

The binding of the pentaene antibiotic filipin to egg-yolk phosphatidylcholine (EPC) and dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles, has been studied by ultraviolet (UV) absorption and circular dichroism (CD). A stoichiometry of one molecule of filipin for five molecules of phospholipid was demonstrated by CD when phospholipids were in fluid phase. The similarity of the CD spectra with EPC and DMPC established a similar filipin-phospholipid assemblage in both membranes. We therefore postulated that filipin incorporation leads to the formation of gel-like domains in fluid EPC membranes as previously demonstrated for fluid DMPC membranes (Milhaud, J., Mazerski J., Bolard, J. and Dufoure, E.J. (1989) Eur. Biophys. J. 17, 151-158). The release of fluorescent probes (carboxyfluorescein (CF) or calcein (CC)), entrapped in EPC small unilamellar vesicles (SUV), due to the action of filipin, was followed by fluorescence and CD measurements concomitantly. The following observations were made. (1) The percentage of released probe, as a function of the filipin/phospholipid molar ratios, was the same whether or not membranes contained cholesterol. (2) The permeabilization of vesicles proceeded concomitantly with filipin-phospholipid binding while filipin-cholesterol binding leveled off. (3) The release of the content of vesicles occurred by an all-or-none mechanism leaving the depleted vesicles intact. From these observations and from the previous structural findings, a new interpretation of the action of filipin is proposed. Precluding any disruptive effect, inducement of permeability would result from the high intrinsic permeability of the interfacial region at the boundaries of the gel-like domains corresponding to the filipin-phospholipid aggregates. Additionally, we obtained the permeability coefficients for the anionic forms of CC and CF across EPC SUV, 0.6.10(-10) cm s-1 and 2.10(-10) cm s-1, respectively, as compared to 2.5.10(-14) cm s-1 for the counterion Na+ (Hauser, H, Oldani, D. and Phillips, M.C. (1973) Biochemistry 12, 4507-4517).     

Correlation between spermine stimulation of rat liver Ile-tRNA formation and structural change of the acceptor stem by spermine

We have recently reported that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation [Peng, Z. et al. (1990) Arch. Biochem. Biophys. 279, 138-145]. To pinpoint which interaction of spermine is more important for spermine stimulation of Ile-tRNA formation, Ile-tRNA formation and ribonuclease V1 sensitivity of tRNA(Ile) were studied using purified tRNAs(Ile) from rat liver, wheat germ, brewer's yeast, torula yeast and Escherichia coli. The results indicate that spermine stimulation of rat liver Ile-tRNA formation correlated with the structural change of the acceptor stem by spermine. The nucleotide sequence of wheat germ tRNA(Ile) was also determined.     

Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde

o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.     

Pre-steady-state properties of bovine heart mitochondrial ATPase: a nucleotide-dependent H+ burst

The transient kinetics of bovine heart mitochondrial ATPase (F1) depleted of loosely bound nucleotides were observed. The activation process which was shown as a lag time before steady-state hydrolysis observed previously (Clark et al. (1984) Arch. Biochem. Biophys. 233, 378-392) was preceded by a proton burst when F1 was stripped of its loose nucleotides. 5'-Adenylylimidodiphosphate (Ado PP[NH]P) or MgATP binding is shown to cause proton release. maximum proton release per F1 free of loosely bound nucleotides is observed with MgATP. Modification with NBD-CL of F1 that was nucleotide-depleted eliminated the proton burst, which suggests that the modified tyrosine (i.e., in the catalytic subunit) is directly involved in the release of protons.     

Filipin as a cholesterol probe. I. Morphology of filipin-cholesterol interaction in lipid model systems

We report some novel morphological observations on the interaction of the polyene antibiotic filipin (crude complex) with cholesterol, studied in non-cellular systems with replication, freeze-fracture, and negative stain techniques. Cholesterol crystals, lecithin liposomes containing 0 to 20 mole% of cholesterol, and liposomes containing 10 mole% of cholesterol and 5 to 40 mole% of sphingomyelin were incubated for varying lengths of time with filipin at different cholesterol: filipin molar ratios. The resulting filipin-induced lesions (FIL) were pleomorphic in all systems studied. In replicas of crystals, FIL appeared as ridges which were either straight, or curved into C- and S-shaped figures or closed circles. Negatively stained preparations showed FIL as white lines of the same configurations and in addition revealed a delicate veil attached to individual FIL. FIL, fused by their veils into clusters or large sheets ("holey sheets"), were shed from crystals. Incubation of liposomes for 1 h at cholesterol:filipin molar ratios of 4:1, 2:1, 1:1, and 1:5, demonstrated that cholesterol detection (i.e. formation of FIL) depend upon the ratio of cholesterol to filipin. At a 1:1 molar ratio FIL formed on liposomes containing 10 mole% cholesterol or more, but detectability increased to 5 mole% at the 1:5 ratio. Increasing the molar ratio of cholesterol:filipin to 2:1 and 4:1 decreased cholesterol detectability to between 10 and 20 mole%. Increasing concentrations of sphingomyelin decreased cholesterol detectability at the 1:1 cholesterol:filipin ratio; further, FIL in sphingomyelin-containing liposomes tended towards larger diameters. Filipin induced aggregation of liposomes and linked them together by holey sheets, providing evidence for filipin-induced extraction of cholesterol from liposomes. Taken together our morphological observations on filipin-cholesterol interaction in non-cellular systems raise pertinent questions as to the feasibility of filipin as a cholesterol probe in cellular systems.     

Hormonal effects on mitochondrial respiration: potential role of endogenous lipolytic activities

Hormonal effects on heart mitochondrial metabolism are investigated by comparing respiratory rates, Ca2+ uptake capacity, and lipolytic activities of mitochondria isolated from control rats to those of mitochondria isolated from thyroparathyroidectomized animals. Two biochemically and morphologically distinct populations of heart mitochondria are prepared--one derived from the region of the cell directly beneath the sarcolemma (subsarcolemmal mitochondria), the other originally between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria isolated from normal rat cardiac tissue have both lower respiratory rates and Ca2+ uptake capacity than do interfibrillar mitochondria. However, when these mitochondrial populations are isolated from hearts from thyroparathyroidectomized rats, there is a selective increase in the maximal ability of the subsarcolemmal mitochondria to accumulate Ca2+, which is accompanied by a proportionate increase in their maximal respiratory rates. Neither Ca2+ uptake capacity nor respiratory rates are similarly increased in the interfibrillar mitochondria. Cytochrome contents and mitochondrial protein recoveries are not significantly changed in either of these mitochondrial preparations. The relationship between these selective increases in respiratory properties of the subsarcolemmal mitochondria to endogenous lipolytic activities is also investigated. It was previously demonstrated that, in the absence of Ca2+, both the rate and extent of formation of free fatty acids from endogenous phospholipids is greater in subsarcolemmal than interfibrillar mitochondria (J. W. Palmer et al. (1981) Arch. Biochem. Biophys. 211, 674-682). In this study it is shown that lipolysis is also more sustained in the subsarcolemmal mitochondria when Ca2+ is added. In the subsarcolemmal mitochondria isolated from thyroparathyroidectomized rats, however, the rates of release of stearic acid and oleic acid are reduced in both the presence and absence of Ca2+. In the presence of added Ca2+, the rate of release of arachidonic acid is also decreased compared to control subsarcolemmal mitochondria, suggesting that the expressed activity of Ca2+-activated phospholipase A2 is lower in those mitochondria isolated from the thyroparathyroidectomized animals, in which respiratory rates and Ca2+ uptake capacity are increased.     

Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes.

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.     

Evidence for a family of four immunochemically related isozymes of cytochrome P-450 purified from untreated rats

Hepatic microsomal cytochromes P-450f, P-450g, P-450h, and P-450i have recently been purified to electrophoretic homogeneity from untreated adult rats and identified as distinct isozymes [Ryan et al. (1984) J. Biol. Chem. 259, 1239-1250; Haniu et al. (1984) Arch. Biochem. Biophys. 235, 304-311]. In Ouchterlony double-diffusion plates, as well as enzyme-linked immunosorbent assays and Western blots, purified immunoglobulin G (IgG) raised in rabbits against cytochromes P-450f or P-450g show strong cross-reactions with the heterologous protein, indicating apparent partial identity. Anti-P-450f and anti-P-450g also show strong cross-reactivity with cytochromes P-450h and P-450i but not with five previously characterized rat hepatic cytochromes, P-450 (i.e., P-450a-P-450e), indicating a high degree of immunochemical and structural relatedness among cytochromes P-450f, P-450g, P-450h, and P-450i.     

Characterization of the collagen chains synthesized by cultured smooth muscle cells derived from rhesus monkey thoracic aorta.

Five different collagen chains and one smaller collagenous fragment have been isolated from the collagens found in the combined cell layer and medium of rhesus monkey aortic smooth muscle cell cultures. The collagen chains which can be identified are alpha1 (III), alpha1(I), alpha2, A and B. The smaller collagenous peptide exhibits an apparent molecular weight of 45 000 and has been designated CP45 (Mayne, R., et al. (1977), Arch. Biochem. Biophys. 181, 462). Smooth muscle cells continue to synthesize the collagens from which these components are derived for at least eight passages in culture. At each passage the alpha1 (III) chain consistently represents about one-half of the total collagen which is recovered after initial fractionation by agarose gel chromatography. The results show that smooth muscle cells derived from rhesus monkey thoracic aorta are phenotypically stable for many generations in vitro.