Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli

Abstract The genes oadGAB encoding the oxaloacetate decarboxylase γ, α and β-subunits from Klebsiella pneumoniae were expressed in Escherichia coli . Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed α-subunit or a combination of a and β-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a γα-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the γα-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the a-subunit was not completely complexed to the membrane-bound γ or βγ-subunits.     

Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

Pyruvate carboxylase in Leptosphaeria michotü. Isolation, properties, intracellular localization and cyclic variations of its activity in relation to polypeptide level

Pyruvate carboxylase (EC 6.4.1.1) was obtained from the fungus Leptosphaeria michotü (West) Sacc. and enriched 543-fold by a 5-step purification procedure as an a4-β4 tetramer of M_r 440000, composedof a M_r 60000 α-subunit, containing bound biotin, and a M_r 50000 β-subunit. The enzyme was active from pH 6.5 to 12.0, with a maximum between pH 8.0 and 8.5. Its specific activity was 125nkat (mg protein)⁻¹: it was not affected by acetyl CoA. A rabbit antiserum raised against the yeast pyruvate carboxylase was specifically reactive against the α-subunits of the L. michotü enzyme. The enzyme was localized into the cytosol by gold-labelled streptavidin and immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Pyruvate carboxylase and acetylCoA carboxylase in L. michotü had synchronous activity rhythms at constant temperature and in darkness; these rhythms were suppressed by cycloheximide or avidin supply. The pyruvate carboxylase level was quantified along the activity rhythm by gel electrophoresis using ³⁵S-streptavidin. and by enzyme-linked immunosorbent assay (ELISA) using serum against the yeast pyruvate carboxylase. The cyclic variations of pyruvate carboxylase activity were correlated with cyclic variations in the enzyme level. Suppression of pyruvate and acetyl CoA carboxylase activities by avidin had a no important effect on the transaminase rhythms of L. michotü .     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Isolation and characterization of oxaloacetate decarboxylase of Salmonella typhimurium,a sodium ion pump

Anaerobic growth of Salmonella typhimurium on citrate is Na⁺-dependent and requires induction of the necessary enzymes during a 20–40 h lag phase. The citrate fermentation pathway involves citrate lyase and oxaloacetate decarboxylase. The decarboxylase is a membrane-bound. Na⁺-activated, biotin-containing enzyme that functions as a Na⁺ pump. Oxaloacetate decarboxylase was isolated by affinity chromatography of a Triton X-100 extract of the bacterial membranes on avidin-Sepharose. The enzyme consists of three subunits , , , with apparent molecular weights of 63800, 34500 and 10600. The -chain contains a covalently attached biotin group and binds to antibodies raised against the -subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae. The Na⁺ transport function was reconstituted by incorporation of the puriried enzyme into proteoliposomes.     

The Characterization of the α-Subunits of 7S Nerve Growth Factor

Abstract: The molecular weight of the α-subunit from 7S Nerve Growth Factor was found to be 26,500 by sedimentation equilibrium and by electrophoretic analysis under denaturing conditions of the native and dimethylsuberimidate cross-linked α-subunit. The α-subunit, and each of the individual α-subunits in this preparation, contained one heavy peptide chain of molecular weight 17,200 and one light peptide chain of molecular weight 9500, held together by disulfide bridges. It is likely that the charge differences between the individual α-subunits arise from posttranslational modifications of a parent α-subunit.     

Heterogeneity of neuronal nicotinic acetylcholine receptors: Structural and functional aspects

Decay kinetics of the postsynaptic excitatory currents (EPSC), distribution of the antibodies specific to different α-subunits of neuronal nicotinic acetylcholine receptors (nAChR), and the effects of these antibodies on ACh-induced membrane currents were studied in neurons of different autonomic ganglia of rats. It was shown that α3-, α5- and α7-subunits were present in all studied cultured neurons of the rat superior cervical ganglion (SCG), while the α4-subunit was present only in about half of the neurons; this α-subunit distribution differed from that in cultured intracardial neurons of rats. Two nAChR populations were found in rat SCG neurons, and a series of nAChR populations were found in murine superior mesenteric ganglion neurons; they differed in kinetics of their ion channel activity, voltage dependence and the rate of their open channel blockade. The possible functional role of neuronal nAChR heterogeneity is discussed.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Anaerobic growth of Salmonella typhimurium on l(+)- and d(−)-tartrate involves an oxaloacetate decarboxylase Na+ pump

We show here that the Enterobacterium Salmonella typhimurium LT2 has the capacity to grow anaerobically on l(+)- or d(-)-tartrate as sole carbon and energy source. Growth on these substrates was Na⁺-dependent and involved the l(+)- or d(-)-tartrate-inducible expression of oxaloacetate decarboxylase. The induced decarboxylase was closely related to the oxaloacetate decarboxylase Na⁺ pump of Klebsiella pneumoniae as shown by the sensitivity towards avidin, the location in the cytoplasmic membrane, activation by Na⁺ ions, and Western blot analysis with antiserum raised against the K. pneumoniae oxaloacetate decarboxylase. Participation of an oxaloacetate decarboxylase Na⁺ pump in l(+)-tartrate degradation by S. typhimurium is in accord with results from DNA analyses. The deduced protein sequence of the open reading frame identified upstream of the recently sequenced oxaloacetate decarboxylase genes is clearly homologous with the -subunit of l-tartrate dehydratase from Escherichia coli. Southern blot analysis with S. typhimurium chromosomal DNA indicated the presence of probably more than one gene for oxaloacetate decarboxylase.     

GABAA Receptors Containing the α4-Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ

Abstract: Recombinant GABA_A receptors, expressed from α-, β-, and γ2-subunits, are diazepam-insensitive when the α-subunit is either α4 or α6. In situ, diazepam-insensitive receptors containing the α6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABA_A receptors in various brain areas containing the α4-subunit. GABA_A receptors immunoprecipitated with a newly developed α4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2-recombinant receptors and diazepam-insensitive [³H]Ro 15-4513 binding sites in rat brain membranes. In addition, α4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4-subunit containing receptors revealed that the α4-subunit contributes to several receptor subtypes. Depending on the brain region, the α4-subunit can be coassembled with a second type of α4-subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [³H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.     

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.     

Localisation of the active site of pyruvate carboxylase by electron microscopic examination of avidin-enzyme complexes

Negatively stained enzyme-avidin complexes, seen at different stages of the titration of the biotin-binding sites on avidin with chicken liver pyruvate carboxylase, have been studied using electron microscopy. Formation of linear, unbranched polymers of the enzyme-avidin complex is seen to occur when the ratio of avidin to enzyme is between 2:1 and 1:2; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron-like structure, observed in the single molecules, has been preserved. From the structure of the polymers and the observation of single enzyme-avidin complexes, we deduce that the biotin groups on the enzyme are located on the external faces of each subunit close to, and probably within 3 nm of, the intersubunit junction.     

Properties of oxaloacetate decarboxylase from Veillonella parvula.

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.     

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.     

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

The mechanism of oxaloacetate decarboxylase of Klebsiella aerogenes was investigated by enzyme kinetic methods. The activity of the decarboxylase was strictly dependent on the presence of Na+ or Li+ ions. For Li+ the Km was about 17 times higher and the Vmax about 4 times lower than for Na+. No activity was detectable at Na+ concentrations less than 5 microM. The curve for initial velocity versus Na+ concentration was hyperbolic. Initial velocity patterns with oxaloacetate or Na+ as the varied substrate at various fixed concentrations of the cosubstrate produced a pattern of parallel lines which is characteristic for a ping-pong mechanism. Product inhibition by pyruvate was competitive versus oxaloacetate and noncompetitive versus Na+. Oxalate, a dead-end inhibitor, was competitive versus oxaloacetate and uncompetitive versus Na+. The inhibition patterns are not consistent with a ping-pong mechanism comprising a single catalytic site but are analogous to kinetic patterns observed with the related biotin enzyme transcarboxylase, for which a catalytic mechanism at two different and independent sites has been demonstrated. The kinetic and other data support an oxaloacetate decarboxylase mechanism at two different sites of the enzyme with the intermediate formation of a carboxybiotin-enzyme complex. The first site is the carboxyltransferase which is localized on the alpha chain and the second site is the carboxybiotin-enzyme decarboxylase which is probably localized on the beta and/or gamma subunit. Binding studies with oxalate indicated that this is bound with high affinity to the alpha chain. The affinity was not affected by Na+ or by complex formation with the beta and gamma subunits. Oxalate protected the decarboxylase from heat inactivation but not from tryptic hydrolysis. The carboxybiotin-enzyme intermediate prepared from oxaloacetate decarboxylase with high specific activity was rapidly decarboxylated in the presence of Na+ ions alone. The effect of pyruvate on this reaction, noted previously, probably results from inhomogeneity of the enzyme preparation used which contained a considerable amount of free alpha subunits.     

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.     

A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp⁵⁹⁰ and Tyr⁶²⁸ and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.     

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila

Abstract Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the α-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae . The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oad A⁻ mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oad A gene product has an effect on the intracellular survival of L. pneumophila .     

Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.     

Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye

The Drosophila genome contains at least three loci for the Na,K-ATPase β-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase β-subunit, as its protein product co-precipitates with the Na,K-ATPase α-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the α-subunit and of the β-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase β-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major β-subunit; the visual cells R1–R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, β-subunits do not co-distribute exactly with the α-subunit at some developmental stages, supporting the concept that the α-subunit and β-subunit can exist in the plasma membrane without being engaged in α/β heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the α-subunit to septate junctions throughout development.