A new purification procedure and molecular properties of Pseudomonas cytochrome oxidase

A procedure has been developed for purification of the cytochrome oxidase from Pseudomonas aeruginosa (EC 1.9.3.2) using DEAE- and CM-cellulose chromatography, gel filtration and crystallization. The final preparation was found to be homogeneous according to ultracentrifugal and disc electrophoretic criteria. The crystalline preparation also exhibited nitrite reductase activity. The spectrum of the enzyme characterizes it as cytochrome cd. At 280 nm E^{1 %}_{1 cm} was 18.5 after dry weight analysis.

The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° _20,w = 7.36 S, diffusion coefficient D _20,w = 5.36×10⁻⁷ cm²×s⁻¹ and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° _20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis

When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.     

The subunits of human hexosaminidase A.

Previous studies of the subunit structure of hexosaminidase gave ambiguous results, but suggested that the enzyme was composed of six equally sized subunits. Dissociation of hexosaminidase A with p-chloromercuribenzoate produces an alkylated fragment with mol.wt. approx. 50000, which is converted into hexosaminidase S by treatment with dithiothreitol. Treatment of native hexosaminidase A with sodium dodecylsulphate results in the formation of a large and a small fragment. However, although the native enzyme has a sedimentation coefficient of 5.8S, dissociation by S-carboxymethylation and maleic anhydride treatment results in subunits exhibiting a single schlieren boundary on analytical ultracentrifugation with a sedimentation coefficient of 2.18S. These results indicate that the enzyme is composed of four subunits, each with molwt. approx. 25000-27000. The mol.wt. of the native enzymes is calculated to be approx. 110000. Our data are consistent with the subunit structures of hexosaminidases A, B and S as being alpha2beta2, beta4 and alpha4 respectively.     

Subunit dissociation and denaturation of Fasciolaria tulipa hemocyanin

1. The hemocyanin from the marine snail, Fasciolaria tulipa has a molecular weight of 8.6 +/- 0.6 x 10(6) determined by light-scattering and a sedimentation constant of (105.9 +/- 1.1)S. 2. The dissociated subunits at pH 11 and in 8.0 M urea (pH 7.4) had molecular weights of 4.4 x 10(5) and 4.7 x 10(5), close to one-twentieth of the parent didecameric assembly. 3. The pH dependence of the molecular weight profile exhibited bell-shaped transitions in both the presence and absence of Ca2+ and Mg2+ ions. In the physiological pH range of about 7.5-8.2 in divalent ion-containing buffers neither the molecular weight behavior nor the sedimentation patterns suggest any significant dissociation. 4. Both the urea and the Hofmeister salt series were found to dissociate the didecameric hemocyanin assembly. The ureas exhibit increasing effectiveness as dissociating agents with the higher alkyl substituted members of the series, suggesting hydrophobic stabilization of the subunit assembly. 5. Denaturation of the hemocyanin subunits by the urea series follows the same trend in effectiveness as the dissociation reaction; the reagent concentrations required to cause unfolding of the globular domains of the hemocyanin chains were, however, much higher than those needed for dissociation.     

Reaction of yeast phosphofructokinase with succinic and maleic anhydride.

Modification of yeast phosphofructokinase by succinic and maleic anhydride influences the catalytic activity and the allosteric behaviour of the enzyme. Depending on the degree of succinylation and maleinylation a decrease of maximum activity, an increase of the apparent affinity for fructose-6-phosphate, a decrease of the Hill-coefficient and a diminution of ATP-inhibition are observed. Up to about 40% of the lysyl residues could be succinylated without dissociation of the hexameric protein, however with a decrease of the enzyme activity. More extensive succinylation or maleinylation causes a dissociation into subunits. The sedimentation coefficient is lowered from 20 S to about 3 S. The molecular weight of the smallest dissociation product was determined to 50 000 (+/- 10 000) by the sedimentation equilibrium method. The number of bound succinyl groups, as determined from radioactivity incorporation, exceeds the content of lysyl groups of the enzyme, indicating that the modifying reagent is also reacting with other amino acid residues.     

Hemocyanin of the chiton, Stenoplax conspicua (Dall)

1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 X 10(6) daltons, (dt) and a sedimentation coefficient of 60 S. 2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 10(5) and 4.17-4.75 x 10(5) dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly. 3. The pH dependence of the molecular weights from pH 4.5 to 11 exhibit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer. 4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins. 5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+, and analyzed by means of the decamer-dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (Napp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.     

Dissociation of ferritins

Apoferritins prepared from horse spleen and heart and rat heart and liver were dissociated by treatment with acetic acid (pH 1.3-3.0). Sedimentation velocity studies showed that apoferritins of spleen and liver (16-17 S) and heart (18-19 S) dissociated into material sedimenting near 3.2 S. Sedimentation equilibrium measurements determined that most of the material had a molecular weight of 38,000-43,000, corresponding to subunit dimers. Failure to dissociate into subunit monomers was confirmed by gel chromatography on Sephadex G-75 and G-150. With the exception of boiling in sodium dodecyl sulfate, further treatments with 0.1-0.4 M KCl, NaCl, 4-9 M urea, 0.01-0.5 M KSCN, 0.1-0.5% Triton X-100, 5-52% dimethylsulfoxide, 10% ethylene glycol, or 0.1% trifluoroacetic acid all failed to cause dissociation into individual subunits, as did exposure to 6 M guanidine-HCl or formic acid, or prior succinylation and/or nitration of the protein. Reassociation occurred between pH 4 and 7 but was not aided by the addition of Fe(II) or reducing agents. It is concluded that ferritins readily dissociate to subunit dimer units and that further dissociation does not occur without full denaturation of the protein.     

Thiol reduction of human α2-macroglobulin. The subunit structure

1. Human alpha(2)-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s(0) (20,w). 3. The dissociation that occurs in the pH range 4.5-2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.     

Role of MicroRNA Processing in Adipose Tissue in Stress Defense and Longevity

The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner?mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S.?cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of?a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.     

Subunit structure of Achromobacter collagenase.

The highly active form of collagenase (EC 3.4.24.3) from Achromobacter iophagus (specific activity 2 microkat/mg) has a molecular weight of 70,000 and the sedimentation coefficient s20,2 = 4.4 S. It is composed of two subunits of molecular weight 35,000 and s20,w of 2.9 S. The dissociation of the dimer under different conditions resulted in the complete and irreversible loss of enzymic activity. A unique N-terminal sequence Thr-Ala-Ala-Asp-Leu-Glu-Ala-Leu-Val- indicates that the two subunits are identical, at least in the N-terminal part of the polypeptide chain. Reduction and pyridylethylation of the subunit change neither molecular weight nor amino acid composition: therefore each subunit of molecular weight 35,000 consists of a single polypeptide chain. Another active and homogeneous form of Achromobacter collagenase (specific activity 1.64 microkat/mg) gives a value for the apparent molecular weight of 80,000 on sodium dodecyl sulphate-polyacrylamide electrophoresis. It is also a dimer in which each of the two subunits of molecular weight 35,000 binds non-covalently a peptide of molecular weight 5000. The dissociation of this form of collagenase is also accompanied by irreversible loss of enzymic activity. The amino acid composition of the subunits which were isolated from both 70,000 and 80,000 collagenases is the same. The role of dimer-monometer equilibrium in the biological function of collagenase is discussed.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.     

Biogenesis of mitochondria. Two-dimensional electrophoretic analysis of mitochondrial translation products in yeast

1. Mitochondrial translation products of yeast Saccharomyces cerevisiae were separated according to charge as well as molecular weight by a highly resolving two dimensional electorphoretic technique (isoelectric focusing in the first dimension ana SDS-electrophoresis in the second dimension). 2. The major protein components (the oligomeric form of subunit 9 of mitochondrial ATPase, var 1, cytochrome oxidase subunits I, II and III, subunit 6 of mitochondrial ATPase and cytochrome b apoprotein) were identified either from their mobility in SDS-electrophoresis or by using mit- mutants defective in certain mitochondrially made polypeptides. 3. This method allowed the separation of subunit III of cytochrome oxidase and subunit 6 of mitochondrial ATPase which cannot be resolved by conventional SDS-polyacrylamide gel electrophoresis. 4. Subunit II of cytochrome oxiodase resolves in two spots of similar pI values and subunit 6 of mitochondrial ATPase resolves in two spots of similar molecular weight. In both cases the double spots disappear simultaneously following a single mutation in the coresponding structural gene. 5. Total mitochondrial proteins were also resolved two-dimensionally revealing over 100 components. The mitochondrial translation products, with the exception of subunit 9 of mitochondrial ATPase, could be easily recognized among the other mitochondrial proteins.     

Earthworm (Pheretima communissima and Pheretima hilgendorfi) hemoglobins: giant assemblies and subunits

The molecular architecture of hemoglobins and their subunits of the earthworms Pheretima communissima and Pheretima hilgendorfi was investigated. In both species, their s0.20,w of 60.8 S and D020,w of 1.80 . 10(-7) cm2 . s-1 corresponded to a molecular weight of 3.07 . 10(6). From electron microscopic observations, the overall structure of the hemoglobins was shown to be two superimposed hexagonal discs, each composed of six-membered constituents. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of both hemoglobins revealed the presence of five species of subunits with molecular weights of 13,000-14,000 (subunit 1), 27,000-28,000 (subunit 2), 30,000-31,000 (subunit 3), 33,000-34,000 (subunit 4) and approx. 52,000 (subunit 5), respectively, and the molar ratio of these subunits 1:2, 3, 4:5 was 2:3:3. If we consider this set of the subunits 1 to 5 as one unit, the molecular weight of this unit should be 2.7-2.8 . 10(5). This one unit, therefore, should be considered to represent one-twelfth the whole molecule with molecular weight of 3.07 . 10(6).     

Conversion of a mitochondrial precursor polypeptide into subunit 1 of cytochrome oxidase in the mi-3 mutant of Neurospora crassa.

1. The cytochrome-alpha alpha 3-deficient mi-3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41 000-Mr subunit 1 of the wild-type enzyme. An apparent molecular weight of about 45 000 was estimated for the mutant product. 2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase. 3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45 000-Mr polypeptide is converted to a 41 000-Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse-chase labelling kinetics reveal a typical precursor product relationship. 4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase. A possible physiological role of the post-translational processing of the mitochondrially synthesized component is discussed.     

Mechanism of increase in cytochrome c oxidase activity in sweet potato root tissue during aging of slices

The mechanism of an increase in cytochrome c oxidase [EC 1.9.3.1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and antibody to the purified enzyme. 1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide. 2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme a. 3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices. 4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [3H]leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V. The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Hydrodynamic studies on interactions between the components of the liver microsomal cytochrome P-450 system

To understand the different behaviour of cytochrome P-450 systems in kinetics as well as in the demethylase activity, sedimentation and molecular weight experiments have been carried out with the following results: 1) Sedimentation coefficients of solubilized P-450 and P-450 LM2 fractions amount to 24 +/- 4 [S] and 12.8 +/- 1.2 [S], respectively. Molecular weights were determined to be 1.0 +/- 0.2 . 10(6) and 3.0 +/- 0.5 . 10(5) Dalton. 2) Triton N-101 provokes splitting of the associated structure both of solubilized P-450 and P-450 LM2; this effect is reversible. 3) The dissociation depends not only on the absolute concentration of Triton but rather on the Triton P-450 ratio. The dissociation curves of solubilized P-450 and P-450 LM2 are similar in shape and in the Triton/P-450 ratio dependence. 4) In the presence of small concentrations of Triton a more complicated dissociation behaviour was observed with broad integral distribution of the sedimentation coefficients. 5) The ionic detergent cholate splits the associated structure of P-450 LM2 at considerably higher concentrations in comparison with Triton-N 101. 6) Addition of reductase causes a decrease of sedimentation coefficients and molecular weights of solubilized P-450. The same effect in P-450 LM2 could be observed only in the presence of phospholipids.