RESUMO
Among the lytic enzymes secreted by the phytopathogen fungus Sclerotinia sclerotiorum, a starch-degrading enzyme has been isolated and characterized. This glycoprotein of 72 kDa is composed of several isoforms ranging from pI 4.8 to 5.4. The enzymatic parameters have been determined. Specificity studies together with the analysis of the reaction products show that it is an alpha-1,4-glucanohydrolase. This result is also corroborated by the analysis of the N-terminal and two inner amino acids sequences that are very similar to fungal glucoamylase genes or enzymes so far sequenced.
Assuntos
Ascomicetos/enzimologia , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/patogenicidade , Glucana 1,4-alfa-Glucosidase/química , Helianthus/microbiologia , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Amido/metabolismo , Especificidade por SubstratoRESUMO
Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes.
Assuntos
Ascomicetos/enzimologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Ascomicetos/crescimento & desenvolvimento , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Isoenzimas/química , Pectinas , Poligalacturonase/químicaRESUMO
Fourteen forms of endopolygalacturonases were purified from the culture medium of Sclerotinia sclerotiorum with ion exchange chromatographies. Individual forms differ in their isoelectric point but exhibit similar molecular masses. On the basis of the cross-reactions to antibodies raised against a purified acidic endopolygalacturonase, these forms could be divided into two related groups. Polygalacturonase activities constitute a complex enzyme system in which the extensive multiplicity is due to the expression of a large gene family.
RESUMO
1. The presence of glycoproteins within the nucleus of cell is now well established and the question arises on the nature of the nuclear glycosylation and the site of their glycosylation. 2. In order to study endogenous nuclear proteins acceptors, we have isolated a subnuclear fraction: nuclear matrix characterized by DNA, RNA, phospholipids and proteins content. Nuclear matrix acceptors were obtained from nuclei incubated with UDP-N-acetyl [14C]glucosamine. 3. In this report we describe the presence of three major glycoproteins labeled with N-acetyl [14C]glucosamine in the nuclear matrix fraction. We obtained gP32, gP67 and gP70 with pI values around 6.2, 6.5 and 8.2.
Assuntos
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/química , Glicosilação , Ponto Isoelétrico , Masculino , Peso Molecular , Proteínas Nucleares/química , Fosfolipídeos/metabolismo , RNA/metabolismo , Ratos , Ratos Wistar , Ribonuclease Pancreático/metabolismoRESUMO
The non-nuclear membranes and the nuclei prepared from L1210 cells catalyze the in vitro transfer of N-acetyl(14C)glucosamine from UDP-N-acetyl(14C)glucosamine to endogenous glycoprotein acceptors. Adequate analysis of these acceptors have demonstrated that the nucleus has its own N-acetylglucosaminyltransferase system that leads to the formation of N-N'-diacetylchitobiosylated proteins.
Assuntos
Acetilglucosamina/metabolismo , Núcleo Celular/enzimologia , Dissacarídeos , Glicoproteínas/metabolismo , Membranas Intracelulares/enzimologia , Proteínas Nucleares/metabolismo , Animais , Catálise , Eletroforese , Glucanos/metabolismo , Leucemia L1210 , Camundongos , Microscopia Eletrônica , Células Tumorais CultivadasRESUMO
This work demonstrates that (N-acetyl[14C]glucosamine)2 is transferred from dolichyl pyrophosphate-(N-acetyl[14C]glucosamine)2 to endogenous nuclear glycoproteins. The (N-acetyl[14C]glucosamine)2 moiety is N-linked, since it can be released from the tryptic glycopeptides by N-glycosidase F and by hydrazinolysis, but not by beta-elimination. The biological significance of this direct transfer of N,N'-diacetylchitobiose to nuclear proteins remains to be elucidated.
Assuntos
Núcleo Celular/metabolismo , Dissacarídeos , Glucanos/metabolismo , Glicoproteínas/metabolismo , Glicosiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Sequência de Carboidratos , Fosfatos de Dolicol/metabolismo , Glicosilação , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Ratos , Ratos WistarRESUMO
1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate.
Assuntos
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Fígado/metabolismo , Animais , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Receptores Imunológicos/metabolismo , Receptores de N-Acetilglucosamina , Frações Subcelulares/metabolismoRESUMO
Aspergillus niger postmitochondrial fraction, which contains high GTPase activity and high GTP binding capacity, has been subjected to subcellular fractionation on a sucrose gradient. A cytosolic and four membranous populations have been separated according to their relative density. The main difficulty has been the characterization of the plasma membrane of the fungus. This fraction, which does not contain any typical enzyme, has been identified after iodination of the outer proteins of protoplasts from A. niger. The immunological detection has shown the occurrence of cytosolic G proteins and membranous small G proteins located not only in the plasma membrane but also in the membranes of the endoplasmic reticulum.
Assuntos
Aspergillus niger/química , Proteínas de Ligação ao GTP/isolamento & purificação , Membrana Celular/química , Citosol/química , Retículo Endoplasmático/química , Frações Subcelulares/químicaRESUMO
In this report, we describe the main characteristics of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. The glycosylation pathway includes the presence of lipids which mediate the nuclear proteins glycosylation. The level of dolichylphosphate seems low and thus could be a regulation factor in the nuclear glycosylations. The discussion deals with the membranous character of the acceptors and the N-acetylglucosaminyltransferase, the N-linkage of the sugar moiety to nuclear proteins and the function of such glycosylation.
Assuntos
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , N-Acetilglucosaminiltransferases , Proteínas Nucleares/metabolismo , Animais , Bacitracina/farmacologia , Fracionamento Celular , Núcleo Celular/enzimologia , Fosfatos de Dolicol/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Cinética , Metabolismo dos Lipídeos , Fígado/citologia , Membrana Nuclear/metabolismo , Ratos , Ratos Endogâmicos , Tunicamicina/farmacologiaRESUMO
Liver nuclei, prepared from normal and vitamin A-deficient rats, were incubated in the presence of GDP-(14C)mannose or UDP-N-acetyl(14C)glucosamine and the labelled glycoproteins analysed by SDS PAGE. Fluorographic analysis has shown that (14C) mannose labelling is enhanced by vitamin A deficiency whereas N-acetyl(14C)glucosamine transfer remains approximately at the same level regardless of the vitamin A status; we did not notice any modification when the proteins were monitored by Coomassie blue or by silver nitrate.
Assuntos
Acetilglucosamina/metabolismo , Fígado/metabolismo , Manose/metabolismo , Deficiência de Vitamina A/metabolismo , Animais , Peso Corporal , Dieta , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Glicosilação , Fígado/enzimologia , Ratos , Ratos Endogâmicos , Vitamina A/administração & dosagemRESUMO
Male Wistar rats of different vitamin A status (total depletion to moderate deficiency) were administered DDT (5 mg/kg/day) or vehicule (corn oil) i.p. daily for 14 days. Vitamin A-dependent protein mannosylation was measured either by in vivo incorporation of [3H]mannose into liver glycoprotein or by in vitro assay of incorporation of [14C]mannose into mannosylretinyl phosphate. Vitamin A deficiency resulted in a significantly impaired in vivo incorporation of mannose in liver glycoprotein but had no effect on the in vitro transport of mannose via retinyl phosphate. Although DDT induced an increase synthesis of liver proteins in smooth endoplasmic reticulum and caused a diminution of the hepatic vitamin A content, it did not affect vitamin A-dependent protein mannosylation.
Assuntos
DDT/farmacologia , Glicoproteínas/biossíntese , Fígado/metabolismo , Manose/metabolismo , Deficiência de Vitamina A/metabolismo , Vitamina A/farmacologia , Animais , Diterpenos , Fígado/efeitos dos fármacos , Monossacarídeos de Poli-Isoprenil Fosfato/biossíntese , Ratos , Valores de Referência , Frações Subcelulares/metabolismoRESUMO
In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.
Assuntos
Química Encefálica , Coloides , Micelas , Cromatografia em Camada Fina , Detergentes , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Octoxinol , Polietilenoglicóis , SolventesRESUMO
Membrane fusion is a fundamental and wide-spread phenomenon in the functioning of cells. Many studies were carried out concerning fusion of plasma membranes as for example cell-cell fusions or uptake by cells of lipid-enveloped viruses. The present study deals with the interaction of intracellular membranes of Aspergillus niger with artificial membranes (liposomes). Association is monitored by the uptake of radioactive liposomes by fungal microsomal membranes. The discrimination between aggregation and pure fusion is done by layering the liposomes-microsomes mixture on a continuous sucrose gradient. The accurate quantitation of the fusion phenomenon is monitored with a fluorescent assay based on resonance energy transfer (Struck, D.K. et al. (1981) Biochemistry 20, 4093-4099). Both methods show that, at physiological pH, there is a spontaneous fusion of microsomes with cholesterol-free liposomes. This phenomenon is protein dependent as trypsinized microsomal membranes are no longer able to fuse with liposomes. Biological significance of the fusion process has been demonstrated using microsomal intrinsic protein mannosylation assay; the enhancement of the lipid to protein ratio due to the fusion of liposomes with microsomes of A. niger results in an increase in the rate of endogenous proteins mannosylation. Moreover, cytosolic proteins of A. niger promote the fusion of any kind of liposomes with microsomes.
Assuntos
Aspergillus niger/ultraestrutura , Membranas Intracelulares/fisiologia , Lipossomos , Fusão de Membrana , Proteínas/fisiologia , Colesterol/fisiologia , Citosol/análise , Retículo Endoplasmático , Concentração de Íons de Hidrogênio , Manose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , MicrossomosRESUMO
Cow's milk has been shown to contain a protein complex which is able to bind vitamin K1 in a reversible manner. This binding property has been investigated by the celite method which consists in creating a dynamic equilibrium between the adsorbent, the celite and the protein complex for the ligand (vitamin K1). Based on competition experiment, the binding is specific and the vitamin K1 binding protein complex has a molecular weight equal to or higher than 7.5 X 10(2) KD.
Assuntos
Leite/metabolismo , Vitamina K 1/metabolismo , Animais , Ligação Competitiva , Bovinos , Proteínas do Leite/metabolismo , Peso Molecular , Ligação Proteica , Proteínas do Soro do LeiteRESUMO
The presence of a PC-binding activity in the cytosol of Aspergillus niger van Tieghem has been established by measuring the reversible exchange of labeled DPC between an adsorbent (celite) and the cytosol. We have shown that this exchange is dependent upon the temperature and the ionic strength and it varies linearly with the protein concentration. This PC-binding activity is able to discriminate between DPC and some other phospholipids.
Assuntos
Aspergillus niger/metabolismo , Fosfatidilcolinas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Ligação Competitiva , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Terra de Diatomáceas , Concentração Osmolar , Fosfolipídeos/metabolismo , TemperaturaRESUMO
Rat liver microsomes catalyze the transfer of mannose from GDPmannose to both retinyl phosphate and dolichyl phosphate to form mannosylphosphorylretinol, mannosylphosphoryldolichol and GDP. The two reactions differ in term of reversibility. In fact, a 200-fold isotopic dilution of GDP[14C]mannose by unlabeled GDPmannose causes mannosylphosphoryldolichol labeling to disappear almost completely, while mannosylphosphorylretinol labeling remains at the same level. The same observation can be made if the mannose donor is removed by centrifugation and replaced by excess GDP; again mannosylphosphorylretinol is stable, but mannosylphosphoryldolichol drops down to one-third of its initial level, as expected for, respectively, a non-reversible and a reversible reaction. Placed in an aqueous medium, mannosylphosphorylretinol releases mannose 1-phosphate (beta configuration) whereas it is quite stable when kept in a membranous environment. These results strongly suggest that mannosylphosphorylretinol as soon as it is formed is segregated in such a way that it is no longer available to the back-reaction; the functional consequence of this segregation would be the possibility for mannosylphosphorylretinol to mannosylate some non-polar regions of certain protein chains.
Assuntos
Dolicol Monofosfato Manose/metabolismo , Fosfatos de Dolicol/metabolismo , Glicosídeos/metabolismo , Hexosiltransferases/metabolismo , Manosídeos/metabolismo , Manosiltransferases/metabolismo , Microssomos Hepáticos/metabolismo , Açúcares de Poli-Isoprenil Fosfato/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Vitamina A/análogos & derivados , Animais , Compartimento Celular , Diterpenos , Guanosina Difosfato/metabolismo , Masculino , Manose/metabolismo , Ratos , Vitamina A/metabolismoRESUMO
The enzyme properties of a soluble uridine 5'-diphosphate (UDP) glucose: mycosporin-2 glucosyltransferase from spores of Ascochyta fabae Speg. (Fungi imperfecti) were studied. The optimal conditions for the glucose transfer from UDP-glucose to the mycosporin-2 (the amide form being the best acceptor) were determined; for maximal activity the glucosyltransferase requires a pH of about 8.5 and the presence of divalent cations (Mn(2+) being more efficient than Ca(2+) or Mg(2+)). The reaction was not reversible in presence of large amounts of UDP.
RESUMO
Aspergillus niger van Tieghem microsomes contain an enzyme that catalyzes mannose transfer from GDP-mannose to polyprenylphosphate. The studies of the specificity of this enzyme for both the sugar donor (nucleoside diphosphate sugar) and the acceptor (polyprenylphosphates that were made available to the enzyme by means of the fusion of acceptor-loaded liposomes with the microsomal membranes) gave the following results. i) All the polyprenylphosphates from C15 to C120 were acceptors except retinylphosphate. ii) The specificity of the enzyme for both the sugar and the base is very strict.
Assuntos
Aspergillus niger/enzimologia , Hexosiltransferases/metabolismo , Manosiltransferases/metabolismo , Microssomos/enzimologia , Cromatografia em Camada Fina , Cinética , Lipossomos , Fosfatidilcolinas , Fosfatidiletanolaminas , Especificidade por SubstratoRESUMO
The influence of various local anaesthetics has been studied on the mannose transfer enzymic system which is localized in Aspergillus niger van Tieghem microsomal membranes. The n-alkanes and tertiary amines do not seem to react on the first step of the reaction: i.e. the polyprenylphosphate-mannose biosynthesis. However two amines, the dibucaine and to a lesser extent the tetracaine, inhibit the mannose transfer from the polyprenylphosphate-mannose to the endogenous proteins.