CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin

The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.     

Visualization of the large heparan sulfate proteoglycan from basement membrane

Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.     

A small isoform of NADH:ubiquinone oxidoreductase (complex I) without mitochondrially encoded subunits is made in chloramphenicol-treated Neurospora crassa

In mitochondria of Neurospora crassa grown in the presence of chloramphenicol a small form of NADH:ubiquinone reductase is made in place of the normal electron-transfer-complex I. This smaller enzyme has a molecular mass of approximately 350 kDa and consists of (at least) 13 different subunits which are all synthesized in the cytoplasm. The complex I which is normally found in Neurospora has a molecular mass of approximately 700 kDa and consists of around 30 different subunits, of which at least six are made in the mitochondria. Immunoblotting and peptide mapping suggest that the subunits of the small enzyme are homologous to subunits of the large enzyme, one subunit might even be identical. The small and the large NADH:ubiquinone reductases have the same high-affinity binding site for NADH but the two enzymes differ in the affinity and inhibitor sensitivity of the ubiquinone-binding site. The possibility is discussed that the small NADH:ubiquinone reductase is primitive isoform of complex I.     

Paramecium Has Two Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase, One Unique to Cilia

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R₄₄) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R₄₄ were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R₄₈) weakly recognized by one of several monoclonal antibodies against R₄₄, but not recognized by an anti-R₄₄ polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R₄₈ and a 70-kDa form containing C and R₄₄. In the large enzyme, R₄₈ was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R₄₄ The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R₄₈ were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R₄₄, whereas R₄₈ is the R subunit of the large enzyme.     

Identification of an abundant latent 94-kDa gelatin-degrading metalloprotease in human saliva which is activated by acid exposure: implications for a role in digestion of collagenous proteins.

Human saliva was found to contain a latent neutral 94-kDa metalloprotease which degrades denatured collagens. Saliva samples from six normal resting individuals contained an average of 0.34 microgram/ml of latent enzyme. The 94-kDa salivary metalloprotease was found to bind to gelatin and to be immunologically identical to a leukocyte-derived 94-kDa gelatinase/type IV collagenase proenzyme. Exposure of the latent enzyme to acidic conditions (pH 2) followed by neutralization resulted in activation of the proenzyme. The activated enzyme degrades denatured collagens such as gelatin. Since 1-2 liters of saliva is swallowed per day and exposed to gastric acidity, this enzyme could become activated in the gastric compartment and following neutralization in the small bowel, may contribute to the degradation of ingested collagenous proteins.     

Polypeptides and bacteriochlorophyll organization in the light-harvesting complex B850 of Rhodobacter sphaeroides R-26.1

The light-harvesting complex (LHC) B850 from Rhodobacter sphaeroides was dissociated into several fragments by treatment with sodium dodecyl sulfate. The molecular weight of each fragment was determined by using transverse polyacrylamide gel electrophoresis under nondenaturing conditions and gel filtration techniques. Four B850 LHCs were observed, having molecular weights of 60,000, 72,000-75,000, 105,000, and 125,000-145,000, and two small bacteriochlorophyll (Bchl)-polypeptide complexes having molecular weights of 6000-8000 and 12,000-14,000. Each of the B850 complexes contains ca. one Bchl a for each 6.5-kDa protein. The optical absorption and circular dichroism of the B850 LHCs recorded directly from the gels are similar to those measured previously for a 22-24-kDa B850 LHCs by Sauer and Austin [(1978) Biochemistry 17, 2011-2019]. These data, combined with studies of other groups, indicate that the smallest LHC in LH1 and LH2 is a Bchl-polypeptide tetramer. Each tetramer contains two Bchl dimers that probably have the structure of P-860, the primary electron donor in Rhodobacter sphaeroides, and two alpha-beta-polypeptide pairs. Interactions among the paired Bchls shift their individual Qy transitions from 780-800 to 850-860 nm, and interactions among two such pairs induce the circular dichroism signal of the LHCs. Three Bchl-polypeptide tetramers probably form a dodecamer having C3 symmetry, and six such dodecamers organize into a large hexagon that can accommodate one or two reaction center complexes.     

Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.     

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.     

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.     

The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast

In the central reaction of N-linked glycosylation, the oligosaccharyltransferase (OTase) complex catalyzes the transfer of a lipid-linked core oligosaccharide onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). The Saccharomyces cerevisiae OTase has been shown to consist of at least eight subunits. We analyzed this enzyme complex, applying the technique of blue native gel electrophoresis. Using available antibodies, six different subunits were detected in the wild-type (wt) complex, including Stt3p, Ost1p, Wbp1p, Swp1p, Ost3p, and Ost6p. We demonstrate that the small 3.4-kDa subunit Ost4p is required for the incorporation of either Ost3p or Ost6p into the complex, resulting in two, functionally distinct OTase complexes in vivo. Ost3p and Ost6p are not absolutely required for OTase activity, but modulate the affinity of the enzyme toward different protein substrates.     

Characterization of the fragmented forms of calcineurin produced by calpain I

Calcineurin, a calmodulin-stimulated phosphatase from bovine brain, was hydrolyzed by calpain I from human erythrocytes. In the absence of calmodulin, calpain rapidly transformed the 60-kilodalton (kDa) catalytic subunit of calcineurin into a transient 57-kDa fragment and thereafter a 43-kDa limit fragment. In the presence of calmodulin, the 60-kDa subunit was sequentially proteolyzed to a 55-kDa fragment and then a 49-kDa fragment. Upon proteolysis in the absence or presence of calmodulin, the p-nitrophenyl phosphatase activity (assayed in the presence of calmodulin) was increased by 300%. The 43- and the 49-kDa fragments were found to (i) remain associated with the small subunit (17 kDa), (ii) have lost the ability to bind and to be activated by calmodulin, and (iii) have phosphatase activity that was still stimulated by Mn2+ or Ni2+. The 43- + 17-kDa form had similar Km values for various substrates, but the Vmax values were increased compared with the native enzyme. It is proposed that (i) a 43-kDa core segment of the 60-kDa subunit of calcineurin contained the catalytic domain, the small subunit-binding domain, and the metal ion (Mn2+ and (or) Ni2+) binding site; and (ii) two distinct types of inhibitory domains exist near the end(s) of the large subunit, one of which is calmodulin regulated, while the other is calmodulin independent.     

Structure of the complex of yeast adenylate kinase with the inhibitor P1,P5-di(adenosine-5'-)pentaphosphate at 2.6 A resolution

Adenylate kinase from yeast cytosol was crystallized as a 1:1 complex with the inhibitor P1,P5-di(adenosine-5'-)pentaphosphate. The crystalline structure was solved by multiple isomorphous replacement at a resolution of 3 A (1 A = 0.1 nm) and subsequent structural refinement at 2.6 A resolution. The yeast enzyme belongs to the group of large variants among the adenylate kinases, whereas the structurally known porcine cytosolic enzyme is a small variant. A comparison showed that the additional 31-residue segment of the large variants covers the active center. This had not been expected, because small and large variants show similar enzyme kinetics. Apart from this insertion, the chain folds of both adenylate kinases are the same. The yeast enzyme with bound inhibitor, however, assumes a much more closed form. In relation to the porcine enzyme without substrate, a segment of 28 residues containing two helices is rotated by about 30 degrees, closing the deep cleft at the active center. This corresponds to the expected induced fit. Sequence comparisons with other adenylate kinases suggest that one of the adenosine moieties of the inhibitor does not bind at a native nucleotide-binding site of the enzyme.     

Quinol-cytochrome c oxidoreductase from the thermophilic bacterium PS3. Purification and properties of a cytochrome bc1(b6f) complex

A quinol-cytochrome c oxidoreductase (cytochrome bc1 complex) has been purified from plasma membranes of a thermophilic Bacillus, PS3, by ion-exchange chromatography in the presence of Triton X-100. The purified enzyme shows absorption bands at 561-562 nm and 553 nm at room temperature, and 560, 551, and 547 nm at 80 K upon reduction, and gives an ESR signal similar to that of a Rieske-type iron sulfur center. Its contents of protohemes, heme c, and non-heme iron are about 23, 10, and 21 nmol/mg of protein, respectively. The enzyme consists of four polypeptides with molecular masses of 29, 23, 21, and 14 kDa judging from their electrophoretic mobilities in the presence of sodium lauryl sulfate. Since the staining intensities of the respective bands are almost proportional to their molecular masses, the monomer complex (87 kDa) of the subunits probably consists of a cytochrome b having two protohemes, a cytochrome c1 and an Fe2-S2-type iron sulfur center. The 29 and 21 kDa subunits were identified as cytochromes c1 and b, respectively, and the 23-kDa subunit is probably an iron-sulfur protein, since the 14-kDa polypeptide can be removed with 3 M urea without reducing the content of non-heme iron. Several characteristics of the subunits and chromophores indicate that the PS3 enzyme is rather similar to cytochrome b6f (a bc1 complex equivalent) of chloroplasts and Cyanobacteria. The PS3 complex catalyzes reduction of cytochrome c with various quinol compounds in the presence of P-lipids and menaquinone. The turnover number at pH 6.8 was about 5 s-1 at 40 degrees C and 50 s-1 at 60 degrees C. The enzyme is heat-stable up to 65 degrees C.     

Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.     

Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.     

Electron microscopic characterization of helical filaments formed by subunits I and II (core proteins) of ubiquinol: cytochrome c reductase from Neurospora mitochondria

The isolated and water-soluble complex of subunits I and II (core proteins) of ubiquinone:cytochrome c reductase from Neurospora mitochondria forms filaments below pH 6.0. Three independent helical reconstructions of single filaments were compared with the 3-D reconstruction of the native enzyme. A model for the helix is proposed in which the core complex dimers are arranged radially with the face which is proximal to the membrane in the native enzyme on the outside of the helix. The dimension of the core complex dimer perpendicular to the helix axis (70 A) provides an independent estimate of the height of the core complex to that obtained previously from cytochrome reductase crystals. The results of STEM mass measurement and the helical model give a mass per repeating unit of 90 kDa, which would indicate that the monomeric core complex consists of one 45-kDa and one 50-kDa subunit.     

Lipid binding-induced conformational change in human apolipoprotein E. Evidence for two lipid-bound states on spherical particles

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1-191) N-terminal domain and a 10-kDa (amino acids 223-299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment approximately 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (-69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (-39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.     

Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.     

Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat

Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials. (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV. The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550. In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones. Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present. Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions.