Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

Properties of the aromatase system associated with the mitochondrial fraction of human placenta

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35–50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16α-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods.

Rabbit antisera were prepared against cytochrome b5 and NADPH-cytochrome c reductase [EC 1.6.2.4] purified from rat liver microsomes, and utilized in examining the distribution of these and other membrane-bound enzymes among the vesicles of rat liver microsomal preparations by immunoprecipitation and immunoadsorption methods. Smooth microsomes with an average vesicular size of 200 nm (diameter) and sonicated smooth microsomes with an average diameter of 40-60 nm were used in subfractionation experiments. Immunoprecipitation of microsomal vesicles with anti-cytochrome b5 immunoglobulin failed to show any separation of the microsomes into fractions having different enzyme compositions. Cytochrome b5 was apparently distributed among all vesicles even when sonicated microsomes were used. When the antibody against NADPH-cytochrome c reductase was used, however, immunoadsorption of microsomes on Sepharose-bound antibody produced some separation of NADPH-cytochrome c reductase and cytochrome P-450 from NADH-cytochrome b5 reductase and cytochrome b5. The separation was more pronounced when sonicated microsomes were used. These results indicate microheterogeneity of the microsomal membrane, and suggest the clustering of NADPH-cytochrome c reductase and cytochrome P-450 molecules in the membrane.     

The markers of pig heart mitochondrial sub-fractions : I. - The dual location of NADPH-cytochrome c reductase in outer membrane and microsomes.

Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart. A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes. Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450. The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The Km-NADPH were always much higher than those for NADH. In mitochondria the Km for NAD(P)H were dependent on cytochrome c concentration. The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.     

Intermembrane electron transport in the absence of added water-soluble carriers.

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems "intact mitochondria - mersalyzed microsomes" and "mersalyzed mtiochondria - untreated microsomes". No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Participation of a cytochrome b5-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in NADH-semidehydroascorbic acid reductase activity of rat liver

The participation of a cytochrome b₅-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in the NADH-semidehydroascorbate (SDA) reductase activity of rat liver was studied. NADH-SDA reductase activity was strongly inhibited by antibodies against OM cytochrome b and NADH-cytochrome b₅ reductase, whereas no inhibition was caused by anti-cytochrome b₅ antibody. NADH-SDA reductase exhibited the same distribution pattern as OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity among various subcellular fractions and submitochondrial fractions. Both activities were localized in outer mitochondrial membrane. These observations suggest that OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase system participates in the NADH-SDA reductase activity of rat liver.     

Identification of outer mitochondrial membrane cytochrome b5 as a modulator for androgen synthesis in Leydig cells

Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017alpha, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017alpha-catalyzed reactions, 17alpha-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17alpha-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17alpha-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Etude de l'evolution des activites des enzymes mitochondriaux de l'hepatocyte au cours du developpement du rat

Study on the evolution of mitochondrial enzyme activities in hepatocyte during rat development

Some constitutive enzymes of the three isolated fractions of mitochondria outer membrane, inner membrane and matrix, have been investigated in rat hepatocyte during a period varying from the foetal state to the 15th day after birth.

In the three mitochondrial fractions, activities of the studied enzymes present different evolutions. In the matrix, the tricarboxylic enzyme activities have already reached their normal values before birth. In the outer membrane, the NADH-cytochrome c reductase activity increases regularly, in the same way as that of the endoplasmic reticulum NADH-cytochrome c reductase. In the inner membrane, the oxygen consumption is very low before birth, then increases suddenly from the 5th to the 8th day after birth, when it reaches the normal values. The limiting factor of the respiratory chain activities is neither cytochrome oxidase nor the first dehydrogenases.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

Protein sorting between the outer and inner mitochondrial membranes: submitochondrial localization of cytochrome c1 whose presequence is replaced by the amino-terminal region of a 70 kDa outer membrane protein

The amino-terminal region of a 70 kDa mitochondrial outer membrane protein of yeast and the presequence of cytochrome c1, an inner membrane protein exposed to the intermembrane space, are thought to be responsible for localizing the proteins in their final destinations after synthesis in the cytosol. Gene fusion experiments were used to identify signals that are responsible for protein sorting between the outer and inner mitochondrial membranes. The submitochondrial localization of cytochrome c1 whose presequence was replaced by the amino-terminal region of the 70 kDa mitochondrial outer membrane protein has been investigated. We have also used an in vivo complementation assay to determine whether or not a 70k-cyt c1 fusion protein is functional. Both the first half and all of the presequence of cytochrome c1 can be replaced by the amino-terminal 12 or 29 residues of the 70 kDa protein for transport to the inner membrane and functional assembly into succinate-cytochrome c reductase. However, replacements by the amino-terminal 61 residues of the 70 kDa protein result in exclusive localization of the fusion proteins to the outer membrane, and the fusions cannot be assembled into the enzyme complex. These data indicate that a mitochondrial targeting signal alone is sufficient to direct cytochrome c1 of mature size to the inner membrane.     

The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [(14)C]lysine and killed 7.5, 15, 30 and 60min. later. The livers were homogenized in 0.3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4.0 and then with 0.15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [(14)C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0.15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0.15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4.0.     

The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting

Gene fusion experiments were used to identify signals that direct imported precursor proteins to specific intramitochondrial locations in yeast. The amino terminus of alcohol dehydrogenase III (ADHIII, a mitochondrial matrix enzyme) transported attached mouse dihydrofolate reductase (DHFR, a cytosolic enzyme) into the mitochondrial matrix. The presequence of cytochrome c1 (a mitochondrial inner membrane protein protruding into the intermembrane space) transported attached DHFR into the intermembrane space. The first half of the cytochrome c1 presequence, which resembles the ADHIII presequence, is a matrix-targeting sequence: it transported attached DHFR into the matrix. The second half of the cytochrome c1 presequence contains a stretch of 19 uncharged amino acids and may thus be a stop-transfer sequence. We conclude that intramitochondrial sorting involves matrix-targeting and stop-transfer sequences within the cleavable presequence.     

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.