Discovery,structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase

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Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC)

The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a approximately 500-800kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of alpha and beta subunits are at a one:one ratio. The k(cat) value of the recombinant enzyme is determined to be approximately 4.0s(-1). It also possesses K(m) values (ATP: 45+/-11microM; 3-methylcrotonyl-CoA: 74+/-7microM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders.     

Immunocytochemical detection of ribulose bisphosphate carboxylase in capsicum plastids

Ribulose bisphosphate carboxylase (RUBPCase) was localized by fluorescence and gold immunocytochemistry in Capsicum fruits. Chloroplasts of the green fruit are heavily labelled. A positive staining is also obtained with chromoplasts of the ripe rad fruit, but gold labelling is fainter. The presence of reactive RuBPCase in chromoplasts is discussed in relation with the absence of ribosomes in these plastids.     

Mitochondrial targeting signals and mature peptides of 3-methylcrotonyl-CoA carboxylase

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog. The amino-termini containing 39 (MCCalpha) or 20 amino acids (MCCbeta) were both necessary and sufficient for targeting. Structural requirements for mitochondrial import were defined by site-directed mutagenesis. Our studies provide the prerequisite to understand the impact of specific mutations on the clinical phenotype of MCC deficiency.     

Immunocytochemical localization of ornithine decarboxylase in cultured murine cells

Summary Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from ³⁵S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.     

Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

Immunocytochemical localization of indole-3-acetic acid in primary roots of Zea mays

Colloidal gold-labelled antibody was used to localize indole-3-acetic acid in caps of primary roots of Z. mays cv. Kys. Gold particles accumulated on the nucleus, vacuoles, mitochondria, and some dictyosomes and dictyosome-derived vesicles. This is the first localization of indole-3-acetic acid in dictyosomes and dictyosome-derived vesicles and indicates that dictyosomes and vesicles constitute a pathway for indole-3-acetic acid movement in and secretion from root cap cells. Our findings provide cytochemical evidence to support the hypothesis that indole-3-acetic acid plays an important role in root gravitropism.     

Sequence analysis of cDNA encoding phosphoenolpyruvate carboxylase from cultured tobacco cells

The complete nucleotide sequence of cDNA encoding phosphoenolpyruvate carboxylase (PEPCase) from cultured tobacco (a C₃ plant) cells was determined and the deduced amino acid sequence was compared with those of PEPCases from other higher plants.     

Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthetic gas-exchange characteristics in ears of Triticum durum Desf.

The presence and distribution of phosphoenolpyruvate carboxylase (PEPCase) in the glumes and immature grains of durum wheat (Triticum durum Desf.) were studied by electron-microscopical immunolabeling of PEPCase with polyclonal antibodies followed by protein A-gold. Plants were grown under mediterranean field conditions and samples were obtained two weeks after anthesis. In the kernels, high gold label was associated with the unstained areas of the protein bodies of aleurone cells, whereas labeling in the cytoplasm and chloroplasts of the pericarp was slight, although significantly above the background. In the glumes, high gold label was only located in cytoplasmic granules (vesicles) of the mesophyll cells, although labeling in the cytoplasm and chloroplasts was also significantly above the background. These observations in immature kernels and glumes are in accordance with the anaplerotic role of this enzyme, as evidenced in C₃ plants. Measurements of apparent photosynthesis and its O₂ dependence and CO₂ compensation concentration were made on ears and flag leaves of durum wheat. In addition, an analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxy-phosphinoylmethyl-2-propenoate, was used to inhibit PEPCase and, thereby, to assess the contribution of the PEPCase to photosynthesis in detached ears. There was no effect of the inhibitor on the apparent photosynthesis of ears. Whereas inhibition of apparent photosynthesis by 210 mL · L^–1 O₂ in flag leaves was typical of C₃ species, inhibition in ears was even greater. The CO₂ compensation concentrations in different ear parts were similar to or higher than in flag leaves and the O₂ dependence was also comparable (about 70%). Therefore, gas-exchange data give further support to the assumption that a C₄ cycle is absent or limited to very low rates in ears of durum wheat.Abbreviations and Symbol AP apparent photosynthesis - BSA bovine serum albumin - DCDP 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate - PBS phosphate-buffered saline - PEP phosphoenolpyruvate - PEPCase PEP carboxylase - CO₂ compensation concentration We thank Dr. Sam Sun (University of Hawaii) for kindly providing the PEPCase antibodies and Drs. C.L.D. Jenkins and M.D. Hatch (Division of Plant Industry, CSIRO, Canberra, Australia) for supplying DCDP for these experiments. We are also grateful to Dr. Robert Bargalló and Mrs. Ana Ribero (Servei de Microscopia Electronica, Universitat de Barcelona) for technical assistance in electron-microscopy studies. Participation of Drs. J.L. Araus and J. Bort in this work was supported by the Research Project of CICYT AGF92-0301, Spain.     

Immunocytochemical localization of laminin in rat anterior pituitary cells in vivo and in vitro

Summary The distribution of laminin was investigated by immunocytochemistry in the rat anterior pituitary in vivo and in primary culture. It was localized by immunofluorescence and by immunoperoxidase in the basement membranes of the pituitary in vivo. In addition it was also found inside glandular cells both in vivo and in culture. The number of immunoreactive cells greatly varied depending on the technical approach used. It was always higher in primary cultures than in vivo. At the electron microscope level, a staining was observed on secretory granules, on rough endoplasmic reticulum cisternae as well as on the membrane of some Golgi saccules and vesicles. Such a localization, at the level of subcellular sites involved in the secretory process, suggests that these cells are able to synthesize and to export in vivo as well as in vitro this component of their basement membranes. This work was supported by grants from CNRS (Grant E.R. 89 and ATP “Pharmacologie des Récepteurs des Neuromediateurs”). Part of this work was performed at the EMBL (Heidelberg) during a short stay of C. Tougard (supported by an EMBO short term fellowship). EDITOR'S STATEMENT This paper documents the interesting observation that glandular cells from anterior pituitary contain laminin in their basement membranes and also apparently synthesize and secrete this extracellular matrix component. Gordon H. Sato     

Distribution and fine structure of ependymal cells possessing intracellular cysts in the aqueductal wall of the rat brain

Summary The wall of the cerebral aqueduct was examined in 20 male rats at the light- and electron-microscopic levels. Disorder in ciliary orientation was occasionally seen in ordinary ependymal cells. Ependymal cells possessing intracellular cysts of 5 to 30 urn in diameter were observed within and beneath the aqueductal ependyma in all animals examined. Light-microscopic reconstruction from serial, 10-m thick frontal sections revealed an extensive distribution of cystic ependymal cells (CECs), especially along the ependymal ridges in the rostral half of the aqueduct, and along the dorsal region of the aqueductal lining in the caudal half. Both cystic and surface membranes of CECs bore microvilli and cilia. Ectopic ependymal cells (EECs) characterized by densely packed microvilli, well-developed intermediate junctions and cilia, but without cysts, were situated in the subependymal region adjacent to a CEC or another EEC. The ependymal ridges were long, narrow and sporadically stratified ependymal linings extending rostrocaudally and bilaterally along the aqueductal surface. Tanycyte-like cells filled the surface region of the ridge, and CECs and EECs were frequently seen in the core. Intraventricularly injected microperoxidase was detected among densely packed microvilli but not in the cystic lumina of CECs, indicating that EECs and CECs are distinct entities.     

Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells

The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK phosphorylation and fatty acid oxidation were measured. A 2-fold increase in both AMPK and ACC phosphorylation was observed in the presence of palmitate concentrations as low as 10 microM, which was also accompanied by a significant increase in fatty acid oxidation. The effect of palmitate on AMPK and ACC phosphorylation was dose-dependent, reaching maximum increases of 3.5- and 4.5-fold, respectively. Interestingly, ACC phosphorylation was coupled with AMPK activation at palmitate concentrations ranging from 10 to 100 microM; however, at concentrations >200 microM, ACC phosphorylation and fatty acid oxidation remained high even after AMPK phosphorylation was completely prevented by the use of a selective AMPK inhibitor. This indicates that LCFAs regulate ACC activity by AMPK-dependent and -independent mechanisms, based on their abundance in skeletal muscle cells. Here, we provide novel evidence that the AMPK/ACC pathway may operate as a mechanism to sense and respond to the lipid energy charge of skeletal muscle cells.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.     

Cell-cycle-dependent cortical localization of pEg3 protein kinase in Xenopus and human cells

BACKGROUND INFORMATION: Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR-1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell-cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell-cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells. RESULTS: By expression of GFP (green fluorescent protein)-tagged pEg3 and indirect immunofluorescence with specific antibodies, pEg3 was found to be localized in the cytoplasm and the nucleus in interphase cells. During mitosis pEg3 was also found in the cytoplasm. From anaphase to telophase, a proportion of the protein was detected at the cell cortex. The cortical distribution in mitotic cells was dependent on F-actin, because the actin-depolymerization-inducing drugs cytochalasin D or latrunculin A prevented pEg3 cortical localization. The protein lacking the conserved C-terminal domain was not detected at the cell cortex, whereas the C-terminal domain was targeted to the cell periphery. In contrast with full-length pEg3, the cortical localization of the C-terminal domain and construct lacking the N-terminal domain was cell-cycle independent, and these constructs were found at the cell periphery in interphase cells. CONCLUSIONS: pEg3 is localized at the cell periphery specifically during mitosis. The C-terminal domain is the only pEg3 domain found to be necessary and sufficient for cortical targeting. Cortical distribution of pEg3 also requires the F-actin cytoskeleton. The cell-cycle-independent cortical localization of the pEg3 C-terminal domain and a construct lacking the N-terminal domain indicates that a negative control mechanism involving the pEg3 catalytic N-terminal domain probably acts to prevent pEg3 cortical distribution during interphase. These results suggest that pEg3 might play a role at the cell cortex during mitosis.     

Plants contain multiple biotin enzymes: discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom

Acetyl-CoA carboxylase is the sole biotin enzyme previously reported in plants. Western analysis with 125I-streptavidin of proteins extracted from carrot somatic embryos visualized six biotin-containing polypeptides, the relative molecular masses of which are 210,000, 140,000, 73,000, 50,000, 39,000, and 34,000. This multiplicity of the biotin-containing polypeptides can be partly explained by the discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase in extracts of somatic carrot embryos, biotin enzymes previously unknown in the plant kingdom. These biotin enzymes seem to be widely distributed in the plant kingdom.     

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 .     

Immunocytochemical localization of arabinoxylans in the cell wall of maize apical internode after microbial degradation in the rumen

Summary— Arabinoxylans were localised by immunocytochemistry using polyclonal antibodies in the cell walls of the apical internode of maize after degradation in the rumen. In order to understand the significance of arabinoxylan in digestibility property, two lines of maize differing in digestibility were used. Wide variations in the intensity of labelling were observed in the four tissues studied (sclerenchyma, fibres, xylem and parenchyma) from the first hours of incubation in the rumen. Incubation time in the rumen greatly influences the intensity of labelling.