Pyruvate kinase functions in hot and cold organs of tuna

Summary The skipjack tuna maintains its red skeletal musculature well above ambient temperatures while the temperature of the heart is within 1°C of that of the water. These two tissues exhibit tissue specific forms of pyruvate kinase. The red muscle has one form while the heart has two.TheK _m(PEP) of the red muscle enzymes rises with temperature, within the normal temperature range of the tissue. The affinity of the major form of the heart enzyme for phosphoenolpyruvate is relatively independent of temperature over the physiological temperature range.K _m(ADP) values are temperature independent for both enzymes.Inhibition by alanine of both enzymes is temperature dependent and rises with temperature. The same is true of ATP inhibition of the heart enzyme, but ATP inhibition of the red muscle enzyme is temperature independent. Fructose diphosphate reverses alanine and ATP inhibition at all temperatures.With both enzymes, temperature affects substrate affinities and the sensitivity of the enzyme to metabolite effectors. These effects can be rationalized in terms of physiological significance only in the case of the red muscle enzyme.List of abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - EDTA ethylene diamine tetra acetic acid - FDP fructose 1,6 diphosphate - LDH lactate dehydrogenase - NADH nicotinamide adenine dinucleotide (reduced) - NAD nicotinamide adenine dinucleotide (oxidized) - PEP phosphoenol pyruvate     

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A_1/2 (PLA_1/2) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA_1/2 and acyltransferase activities.     

The ATPMg-dependent phosphatase is present in mammalian vascular smooth muscle

An ATPMg-dependent phosphorylase phosphatase was identified in vascular smooth muscle from bovine aorta. The smooth muscle enzyme, like the corresponding enzyme from striated muscle, exists as an inactive phosphatase (F_C-enzyme) which can be activated by a protein, F_A, in the presence of ATP and Mg²⁺. Moreover, smooth muscle F_C is activatable by skeletal muscle F_A and skeletal muscle F_C can be activated by smooth muscle F_A. The mode of activation of aortic F_C by aortic F_A is similar to that reported for the skeletal muscle proteins. In accord with earlier findings obtained with the skeletal muscle system, the activity of the aortic phosphatase is inhibited by a specific heat-stable modulator protein (previously called phosphatase inhibitor-2). Thus, the fundamental properties of arterial ATPMg-dependent phosphatase appear to be identical to those of its skeletal muscle counterpart which purportedly represents the major phosphorylase phosphatase in that tissue. Since glycogen phosphorylase is activated when vascular smooth muscle contracts, ATPMg-dependent protein phosphatase may participate in coordinating arterial metabolism and contractility.     

Comparative studies on muscle AMP-deaminase--I. Purification, molecular weight, subunit structure and metal content of the enzymes from rat, rabbit, hen, frog and pikeperch.

1. AMP-deaminase (EC 3.5.4.6) from skeletal muscle of frog and pikeperch was purified to homogeneity and compared with the homogeneous enzymes purified from rat, rabbit and hen skeletal muscle. 2. Their molecular weight was close to 280,000, every enzyme consisted of four identical subunits of molecular weight about 70,000. 3. All enzymes were found to contain about two atoms of zinc per molecule. 4. Minor differences of u.v.-absorption spectra between amphibian and fish muscle enzyme as compared with mammalian and bird muscle enzyme were found.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Immunochemical analysis of the peroxisomal beta-oxidation enzymes in rat and human heart and skeletal muscle and in skeletal muscle of Zellweger patients

Immunoblot analyses with antibodies against the peroxisomal beta-oxidation enzymes from rat liver showed the presence of these enzymes in rat and human liver and kidney and rat adrenal gland. The bifunctional protein could not be detected in muscle tissues or cultured muscle cells. Acyl-CoA oxidase was detected in rat heart and cultured human muscle cells. 3-Ketoacyl-CoA thiolase was also detected in human and rat heart and skeletal muscle; however, this enzyme was not detectable in skeletal muscle of Zellweger patients, in agreement with the absence of peroxisomal fatty acid oxidation.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H₂O₂-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of ¹⁴C-labeled acid-soluble products from 1-¹⁴C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H₂O₂ production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.     

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35 000 both before and after 4 M urea treatment. Its activity was specific for the β-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A_0.5 = 0.6 μM). This enzyme had a molecular weight of approx. 70 000 which was reduced to approx. 35 000 after treatment with 4 M urea. It dephosphorylated both the α- and β-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.     

Calmodulin-dependent glycogen synthase kinase: Identification in liver of normal and phosphorylase kinase-deficient rats

We have purified a calmodulin-dependent glycogen synthase kinase from livers of normal and phosphorylase kinase-deficient (gsd/gsd) rats. No differences between normal and gsd/gsd rats were apparent in either (a) the ability of liver extracts to phosphorylate exogenous glycogen synthase in a Ca²⁺- and calmodulin-dependent manner or (b) the purification of the calmodulin-dependent synthase kinase. Although extracts from rat liver, when compared to rabbit liver extracts, had a significantly reduced ability to phosphorylate exogenous synthase, the calmodulin-dependent synthase kinase could be purified from rat liver using a protocol identical to that described for rabbit liver. Moreover, the synthase kinase purified from rat liver had properties very similar to those of the rabbit liver enzyme. The enzyme was completely dependent on calmodulin for activity against glycogen synthase, was unable to phosphorylate phosphorylase b, catalyzed the rapid incorporation of 0.4 mol phosphate/mol of glycogen synthase subunit, selectively phosphorylated sites 1b and 2 in the glycogen synthase molecule, had a Stokes' radius of about 70 Å, and appeared to be composed of subunits of M_r 56,000 and 57,000. These observations led us to conclude that (1) calmodulin-dependent glycogen synthase kinase is distinct from other kinases previously described and (2) the rat liver kinase and the rabbit liver kinase are very similar enzymes.     

The demonstration of a specific 5'-nucleotidase activity in rat tissues.

5′-Nucleotidase has been partially purified from rat liver, spleen, kidney, heart, lung, brain and skeletal muscle. The majority of the enzyme activity in each of these tissues was insoluble in 1% of Triton X-100, solubilized in 2% Triton X-100,1% sodium deoxycholate, and stable to incubation at 50 °C for 5 min. The partially purified enzyme from each tissue exhibited the same pH optimum, was inhibited by concanavalin A, and was inhibited in an identical manner by antibody to highly purified 5′-nucleotidase from liver. Since the enzyme is usually concentrated in the plasma membrane (De Pierre, J. W. and Karnovsky, M. L. (1973) J. Cell Biol., 56, 275–303), the results indicate that the enzyme may represent a convenient and general marker for this organelle in rat tissues.     

Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a M_r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M_r of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and K_a values of 67 nM and 1.1 μM for calmodulin and Ca²⁺, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca²⁺, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

Bovine cytochrome c oxidases,purified from heart,skeletal muscle,liver and kidney,differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.