Novel isolation and reconstitution of the iron-sulfur protein of mitochondrial Complex III

An iron-sulfur protein was isolated from mitochondrial Complex III as a single band on SDS-polyacrylamide gel electrophoresis by hydrophobic interaction chromatography, in which the complex was dissociated into the iron-sulfur protein and the rest of the complex with a loss of enzymic activity. The critical condition for the dissociation was depletion of boundary phospholipids to 0.6% (w/w). After gel filtration of the isolated iron-sulfur protein, 76 ng atom of non-heme iron and 66 nmol of acid-labile sulfide were found per mg of the protein. By incubating the protein with the iron-sulfur protein depleted-complex in the presence of a soybean phospholipid mixture, the enzymic activity was fully recovered. The electrophoretic pattern of the reconstituted complex showed a polypeptide composition similar to that of the original Complex III. These results indicate that the iron-sulfur protein is attached in the complex with the aid of boundary phospholipids.     

Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V–VII were rather resistant to degradation. From the relative inaccessibility of subunits V–VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

An ubiquinone-binding protein in mitochondrial NADH-ubiquinone reductase (Complex I)

An ubiquinone-binding protein (QP) was purified from mitochondrial NADH-ubiquinone reductase (Complex I). Complex I was separated into 3 fragments: a fraction of hydrophobic proteins, that of soluble iron-sulfur protein (IP) and soluble NADH dehydrogenase of flavoprotein by a procedure involving the resolution with DOC and cholate, followed by ethanol and ammonium acetate fractionations. About 40% of the total ubiquinone was recovered in the IP fragment which consisted of 12 polypeptides. The QP was purified from the IP fragment with a hydrophobic affinity chromatography. SDS-polyacrylamide gel electrophoresis showed that the purified QP corresponded to 14-kDa polypeptide of the IP fragment and was a different protein from the QP (12.4 kDa) in Complex III. The purified QP (14 kDa) contained one mol ubiquinone per mol. The ubiquinone-depleted IP fragment could rebind ubiquinone. These results indicate that an ubiquinone-binding site in Complex I is on the 14-kDa polypeptide of the IP fragment.     

Polypeptides in the succinate-coenzyme Q reductase segment of the respiratory chain

Complex II (succinate-coenzyme Q reductase) was resolved into ten different polypeptides by polyacrylamide gel electrophoresis. Four polypeptides, CII-1, CII-2, CII-3, and CII-4 with molecular weights of 70 000, 24 000, 13 500, and 7000, were present in large amounts in all preparations examined. CII-1 and CII-2 are the flavoprotein and iron-sulfur protein, respectively, of succinate dehydrogenase; CII-3 and CII-4 have not been functionally indentified. Six polypeptides were present in much smaller amoumts as judged by staining intensity, and each of these comigrated with components in complex III. The amino acid compositions of several of the minor components in complex II were identical with that of an equivalently migrating polypeptide in complex III. We conclude that succinate-coenzyme Q reductase contains four different polypeptides and is contaminated with variable amounts of complex III when isolated as complex II.     

Detection of iron-sulfur center-containing subunits of mitochondrial NADH dehydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by high-performance gel permeation chromatography

Soluble NADH dehydrogenase resolved from Complex I of the mitochondrial electron-transfer chain was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate at 4 degrees C, and then the gel was stained for iron with bathophenanthroline disulfonate and thioglycolic acid. The 23,000-dalton subunit was markedly stained, and the 51,000-dalton subunit was also stained, but only slightly. High-performance gel permeation chromatography using an eluant containing 0.1% sodium dodecyl sulfate also demonstrated that these subunits contain an iron-sulfur center: the elution pattern recorded by light absorption at 400 nm gave two peaks corresponding to the positions of the subunits.     

Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2.

The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.     

Immunochemical demonstration of the iron-sulfur protein in Complex III of mitochondrial electron-transfer chain

An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.     

Purification of cytochrome b from complex III of beef heart mitochondria

Two cytochrome b preparations have been prepared from Complex III of beef heart mitochondria, by detergent-exchange chromatography on a butyl-Toyopearl column. One was eluted from the column with buffer containing Tween 20 after most of other subunits of Complex III were eluted with buffer containing guanidine-HCl, and the other was eluted from the column with buffer containing sodium dodecyl sulfate. The former is consisted of a single polypeptide (subunit III) and contained 37.5 nmol of heme b/mg of protein, and the latter consisted of subunits III and IX and contained 19.5 nmol of heme b/mg of protein. The former was labile when it was reduced by dithionite, whereas the latter was stable. Subunit IX in the latter is associated with cytochrome b even after gel filtration and density gradient centrifugation. These results suggest that subunit IX plays a role in stabilizing cytochrome b.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Brain UDPgalactose: ceramide galactosyltransferase Purification of a catalytically active protein obtained after proteolytic digestion.

A procedure for the purification of UDPgalactose--2-hydroxyacylsphingosine galactosyltransferase (EC 2.4.1.45) including detergent extraction, ion-excharge chromatography and proteolytic digestion was developed. The active fraction obtained by this procedure had about 100 times higher specific activity than microsomes. Enzymic activity resisted destruction by pronase treatment at 4 degrees C. Agarose gel chromatography indicated the presence of an enzyme-phospholipid-detergent complex with a molecular weight between 400 000 and 500 000. Intact phospholipids seemed to be required for full enzymic activity as evidenced by the drastic loss of activity upon treatment with phospholipase A or C.     

Purification and properties of a virion protein kinase.

The protein kinase associated with virions of frog virus 3 was purified to apparent homogeneity by ion exchange chromatography and gel filtration. The enzyme protein appeared as a single polypeptide of molecular weight 50,000 to 55,000 as determined by gel filtration, glycerol gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and comprised approximately 0.4% of the total virion protein. The activity was classified as a cyclic nucleotide-independent protein kinase as it was not effected by cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, or inhibited by a cyclic nucleotide-dependent protein kinase inhibitor protein, and utilized GTP as well as ATP as a phosphate donor. The greatest rates of phosphorylation were obtained with acidic phosphoprotein substrates such as casein or phosvitin, although potential physiological substrates for this activity included specific virion polypeptides of frog virus.     

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.     

A solid phase assay for galactosyl transferases. Evidence for an active site of less than 14 kDa

Galactosyltransferase (GalTase) prepared from human milk was found to exist as a complex with e-lactalbumin as demonstrated by crossed immunoelectrophoresis against specific antibodies raised against the complex. GalTase activity was stable to proteolysis and, when subjected to gel filtration on Ultrogel AcA54, the enzyme activity eluted as a single peak. A second peak of activity was found to be adsorbed to the column matrix and was eluted with buffer containing 1 M NaC1. The hydrophobic fraction represented 5% of the total GalTase activity in human milk. After polyacrylamide gel electrophoresis the main enzyme activity peak was represented by polypeptides of 67kDa molecular weight and of 14kDa molecular weight. Electroblotting of these peptides onto a nitrocellulose membrane followed by determination of GalTase activity showed activity for 45–55 kDa and for 14 kDa peptides. The hydrophobic fraction from the AcA54 column was resolved into polypeptides of 110 kDa-45 kDa molecular weight, all of which contained GalTase activity after blotting. It is supposed that the GalTase from non-proteolyzed milk is composed of a 14 kDa polypeptide containing the active site together with another part of the polypeptide backbone which is involved in the regulation of GalTase activity by -lactalbumin, a third part of the polypeptide is responsible for the membrane insertion.Abbreviations UDP-Gal uridine diphosphatidyl galactose - GlcNAc N-acetylglucosamine - Glc glucose - PAGE polyacrylamide gel electrophoresis - GalTase galactosyl transferase (EC 2.4.1.22) - -ovo pronosac digest fraction of hen ovomucoid To whom correspondence should be addressed.