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N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.

A deficiency in alpha-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39,000-fold from liver to homogeneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepharose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl alpha-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50 degrees C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of alpha-N-acetylglucosaminidase.

Chlorocruorin was purified from Potamilla leptochaeta and the spectral properties of its derivatives wwere investigated. Ferri- or ferrochlorocruorin did not exhibits a ferrihemochrome or ferrohemochrome spectrum, respectively. Oxy- and carbonmonoxy-ferrochlorocruorin did show ferrohemochrome-type spectra. Ferrihemochromes were formed, however, when oxy-or ferrichlorocruorin was treated with 0.02-0.05% SDS, and they were transformed to ferrohemochromes by reduction with sodium dithionite. Ferrihemochrome formation was also brought about by increasing the pH of a ferrichlorocruorin solution to 9, or by liganding of extrinsic imidazole or cyanide to the ferric pigment. Therefore, it is apparent that at least one of the coordination positions on the heme iron in ferri-and ferrochlorocruorin is vacant or occupied by a weak-field ligand. Titration studies of ferrichlorocruorin with imidazole indicated that this supposedly vacant coordination position was occupied first by the imidazole, and that the intrinsic ligand of protein orgin was replaced finally at higher concentrations. The extrinsic ligands in the cyanide and imidazole complexes of ferrichlorocruorin were excluded from their coordination positions as the protein moiety assumed conformations inherent to the reduced pigment. Spectral analyses indicated that the intrinsic ligand is an imidazole moiety of a histidyl residue. When chlorocruorin was intact, carbonyl reagents such as cyanide and sodium bisulfite did not add to the formyl group of chlorocruoreheme. When the protein conformation was perturbed by SDS, addition to ferrichlorocruorin occurred appreciably. This addition was accelerated if the heme iron coordination position had been occupied by strong field ligands,and was reversed to some extent as the chlorocruorin complexes were reduced.

In dimeric cytochrome oxidase [EC 1.9.3.1], one of the two heme a molecules of one monomeric unit has been proposed to be converted by the other unit, thus becoming latent in terms of catalytic functions (1). As the dimer was split into two monomers by treatment with alkali or sodium dodecyl sulfate (SDS), it was shown that the intensity of circular dichroism (CD) in the Soret region due to heme a decreased, probably reflecting release of the strain on the latent heme. On the other hand, the profile of magnetic circular dichroism (MCD) was nearly unchanged during this conversion, except for a weakening of the signal due to deprotonation of the heme during the alkali treatment. When the monomer was further dissociated into constituent subunits in strong alkali or at high concentrations of SDS, the CD spectrum disappeared almost completely, indicating loss of the asymmetric interactions of the chromophoric heme a with its immediate environments, consisting of the subunit assembly. The MCD pattern also suffered a small change as the dissociation proceeded, and a specific pattern appeared as the Schiff base was finally formed. The Schiff base formation of cytochrome oxidase in strong alkali proceeded in two steps whether the heme iron was in the oxidized or reduced state. As a consequence of the initial rapid reaction, the enzyme was suggested to have been disintegrated into constituent subunits with heme a being attached nonspecifically to either one, and structural characteristics dependent on the redox state were completely lost. The Arrehenius plot for this rapid change showed a break, indicating a transition in the structure of the cytochrome oxidase assembly, although no such phenomenon was observed during the slow reaction. Activation parameters in the rapid and slow reactions for the oxidized and reduced oxidase are given. Based on these findings, as well as other considerations, a molecular architecture of this enzyme is proposed; the role of heme a in anchoring four 14,000-dalton polypeptides into the minimal functional unit catalyzing the aerobic oxidation of ferrocytochrome c is emphasized.

The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.

Addition of NaHSO3 or HCN to the formyl group of heme alpha was greatly accelerated by freezing reaction mixtures prepared in aq. Na2CO3, and freezing resulted in characteristic color and spectral changes of the solutions. Similar changes were observed on decreasing the pH of alkaline reaction mixtures with HCl at room temperature, indicating that the effective pH of certain salt solutions is greatly lowered by freezing. The reactivity of the formyl group changed depending on the redox state of the heme iron and the species of ligand.

Both beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacteriorhodopsin vesicles in the presence of calcium chloride, and the extent of photoactivation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton transport to the reaction center for water formation is the rate limiting step for the cytochrome oxidase activity.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.

Spectrophotometric studies revealed the irreversible photodissociation of reduced cytochrome oxidase-nitric oxide (NO) at 5 K. The dissociated NO recombined as the sample temperature was raised, and the half-recombination temperature was 65 K. The photodissociation at 5K was also confirmed by a change in the EPR spectrum; that is, ferroheme a-NO signals at gx=2.09 and gm=2.006 were replaced by a new signal at gm=2.03, and this change was reversed at the temperature of liquid nitrogen. Comparison of such behavior with that of cytochrome oxidase-carbon monoxide led us to propose that on photodissociation of NO from heme iron, the NO was trapped specifically at a site near the heme iron producing a new paramagnetic species. Its identification will require further studies.

Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome c oxidized/(s X mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14 mM) completely inhibited the peroxidase activity. The inhibition by 1 mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72 mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.

cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.

A method was established to estimate the pH change of several buffers solutions on freezing by using a combination of pH indicators. Among more than 30 buffers solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220 degrees K; the results were tabulated. Glycerol was found to suppress the pH changes because of its "salt buffer" effect.

Dithionite reduced the heme c moiety of Pseudomonas nitrite reductase almost instantaneously, whereas the spectral change of heme d proceeded in two steps, requiring at least 15 min for completion. The final spectrum coincided well with that obtained by anaerobic reduction with ascorbate, during which a quasi oxidation-reduction equilibrium was established between the two heme groups. The difference in apparent redox potential was calculated to be 24 mV, heme d being more negative. When the enzyme was supplemented with a reductant and molecular oxygen, an oxygenated intermediate appeared at the heme d moiety.

The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3). The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C. At liquid nitrogen temperature, CO-rebinding did not occur. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.

In the present paper, the important role played by cytochrome c in the cytochrome oxidase system is demonstrated by using highly purified praparatinos of cytochromes a , c 1 , and c 1 . Despite the fact that cytochrome a can be rapidly reduced by p -phenylene-diamine, the component is not oxidized by oxyge if cytochrome c is not present. The K m calculated for cytochrome c is similar to that found in particulate preparations. The addition of cytochrome c 1 has not effect on the cytochrome oxidase reaction. The reaction between cytochromes c 1 and a is very slow and is mediated by cytochrome c . On the basis of the above results, it is proposed that cytochrome c acts not only as an electron carrier between cytochromes c 1 and a but also as an essential constituent to form an active complex with cytochrome a in the cytochrome oxidase system. The reaction mechanism of teh cytochrome oxidase system is discussed on the basis of the above results.