RESUMO
Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.
Assuntos
Trifosfato de Adenosina/metabolismo , Cílios/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cerebelo/citologia , Cerebelo/metabolismo , Cílios/ultraestrutura , Creatina Quinase Forma BB/genética , Creatina Quinase Forma BB/metabolismo , Expressão Gênica , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glucose-6-Fosfatase/genética , Glicólise , Masculino , Microssomos/metabolismo , Microssomos/ultraestrutura , Neurônios Receptores Olfatórios/citologia , Fosforilação Oxidativa , Ratos , Ratos Sprague-Dawley , Técnicas de Cultura de TecidosRESUMO
One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.
Assuntos
Entamoeba histolytica/ultraestrutura , Microssomos/enzimologia , Proteínas de Protozoários/metabolismo , Acetilglucosaminidase/análise , Fosfatase Ácida/análise , Animais , Western Blotting , Centrifugação com Gradiente de Concentração , Dolicóis/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/fisiologia , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamoeba histolytica/fisiologia , Glucosiltransferases/análise , Glicosilação , Manosiltransferases/análise , Manosiltransferases/genética , Proteínas de Membrana/análise , Microssomos/fisiologia , Microssomos/ultraestrutura , Oligossacarídeos/biossíntese , Complexo de Endopeptidases do Proteassoma/análise , Isomerases de Dissulfetos de Proteínas/análise , Canais de Translocação SECRESUMO
During maturation, rat renal papillary microsomes suffer a rearrangement in their fatty acid phospholipid composition. The most significant changes in total phospholipids are the increase in their content of the 20:4 and a decrease in the levels of 14:0, 16:0, 18:1, 22:6 and 20:3 fatty acids. The changes in total phospholipid fatty acid content are a reflection of the variations in the individual phospholipid composition. During this period, microsomal cholesterol, phospholipid, and protein concentrations present no variations. Steady state fluorescence anisotropy obtained by using TMA-DPH (see text) as a fluorescence probe denoted higher values for 70- versus 10-day-old microsomes. Using DPH as a probe, steady state fluorescence anisotropy was determined in whole microsomes, as well as in total lipid and phospholipid vesicles, from both 10- and 70-day-old papillary cells. No differences were detected in phospholipid and total lipid vesicles between days 10 and 70. On the other hand, 10-day-old microsomes appeared to be less fluid than adult microsomes. The results indicate that these structural changes in kidney membranes during development might affect protein-lipid interaction and, therefore, the activity of many membrane enzymes.
Assuntos
Membranas Intracelulares/fisiologia , Medula Renal/crescimento & desenvolvimento , Medula Renal/ultraestrutura , Fluidez de Membrana , Microssomos/ultraestrutura , Animais , Difenilexatrieno/análogos & derivados , Polarização de Fluorescência , Corantes Fluorescentes , Ratos , Ratos WistarRESUMO
1. The effect of pH on the association of carbonic anhydrase (CA) with bovine gastric light microsomal membranes (LMMs) was investigated (a) by washing LMMs containing CA activity with solutions of different pHs; (b) by studying the adsorption at various pHs of soluble bovine erythrocyte CA to washed gastric LMMs. In both cases, the association of CA with gastric LMMs was dependent on pH, being lower at neutral or alkaline pH. 2. The amount of soluble CA associated with gastric LMMs at pHs 8.0 and 9.0 was reduced when 140 mM K+/10 mM Na+ was added to the incubation medium. 3. Two sources of CA activity in bovine gastric LMMs were assumed: a loosely- and a firmly-membrane-associated activity. Both CA activities were dose-dependently inhibited by acetazolamide (I50: 3.6 x 10(-9) and 8.4 x 10(-9) M, respectively) and by chloride, acetate, iodide, bromide and nitrate at 100 mM. Firmly-membrane-associated activity appeared to be less sensitive to inhibition by acetazolamide, chloride and iodide. 4. Both activities exhibited different behavior and stability following treatment with alkaline Triton X-100. 5. The possible importance of a membrane-associated CA activity in gastric LMMs related to gastric acid secretion is discussed.
Assuntos
Abomaso/enzimologia , Anidrases Carbônicas/metabolismo , Mucosa Gástrica/enzimologia , Abomaso/ultraestrutura , Acetazolamida/farmacologia , Animais , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/isolamento & purificação , Bovinos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/enzimologia , Masculino , Microssomos/enzimologia , Microssomos/ultraestrutura , Octoxinol , Polietilenoglicóis/farmacologia , Potássio/farmacologia , Sódio/farmacologiaRESUMO
1. The occurrence and characteristics of carbonic anhydrase (CA) activity were studied in light microsomal membranes (LMM) purified from bovine gastric mucosa. 2. Bovine gastric LMM contained a high activity of CA ranging from 170 to 400 mumol.H+/min/mg protein when assayed at 0 degree C by pH-stat technique. 3. The addition of 2mM EDTA to the assay mixture increased the enzyme activity. Lower concentrations (0.5-1 mM) had no effect. 4. The enzyme activity was dose-dependently inhibited by acetazolamide and furosemide (I50: 5 x 10(-10) M and 4.8 x 10(-7) M, respectively) and by chloride ion (Ki 85 mM) and appeared to be quite stable to treatment with alkaline Triton X-100. 5. Most of the CA activity is loosely associated with the LMM since it was removed by different washing treatments. Nevertheless, after extensive washes, gastric LMM still contained CA activity suggesting the existence of a firmly membrane-associated form of CA. 6. Values of CA activity higher than those reported previously were found in pig gastric LMM. Furthermore, the washing treatments described in this work were more effective in washing CA activity off pig gastric LMM than procedures described previously.
Assuntos
Anidrases Carbônicas/isolamento & purificação , Mucosa Gástrica/enzimologia , Acetazolamida/farmacologia , Animais , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Bovinos , Cloretos/farmacologia , Ácido Edético/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Furosemida/farmacologia , Mucosa Gástrica/ultraestrutura , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Membranas Intracelulares/enzimologia , Cinética , Masculino , Microssomos/enzimologia , Microssomos/ultraestrutura , Octoxinol , Polietilenoglicóis/farmacologia , Sulfatos/farmacologia , Sulfonamidas/farmacologia , SuínosRESUMO
A microsomal fraction consisting of membranes of transverse tubule origin has been purified by a modification of the calcium-loading procedure initially described by Rosemblatt et al. (J Biol Chem 256:8140-8, 1981). Enzymatic analysis of this fraction shows an enrichment of the vesicles in the Mg++ ATPase (basal) activity characteristic of the T-tubules and an absent or very low Ca++-dependent-ATPase activity. Stereological analysis of freeze fracture replica of the membranes in the purified fraction indicates that they have a very low density of particles in their P faces and lack the structural manifestation of the caveolae typical of the sarcolemma. Immunological analysis performed with monoclonal antibodies prepared against purified T-tubule and sarcoplasmic reticulum membranes define some T-tubule specific antigens and confirm the morphological and biochemical data regarding the origin and purity of the T-tubule preparation.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Músculos/ultraestrutura , Animais , Anticorpos Monoclonais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Imunofluorescência , Técnica de Fratura por Congelamento , Camundongos , Camundongos Endogâmicos BALB C , Microssomos/enzimologia , Microssomos/imunologia , Microssomos/ultraestrutura , Proteínas Musculares/análise , Músculos/enzimologia , Músculos/imunologia , Coelhos , Sarcolema/enzimologia , Sarcolema/imunologia , Sarcolema/ultraestruturaRESUMO
The association of endogenous synenkephalin and met-enkephalin containing peptides with the membrane of bovine chromaffin granules and physicochemical characteristics of this association were studied. The associated materials were only released at a non physiological pH range and this effect was enhanced with growing salt concentrations (0.5, 1.0 and 2.0 M KSCN). A higher peptide dissociation occurred with membrane solubilizing agents (SDS greater than Triton X-100 greater than digitonin). In microsomes the materials dissociated with 2 M KSCN (pH 7.4) corresponded to peptides larger than 12.0 kDa, while in granules corresponded to molecules smaller than 8.5 kDa, displaying synenkephalin and met-enkephalin immunoreactivities. These data suggest that some sequence of the C-terminal portion of synenkephalin may be responsible for the association of proenkephalin derived peptides with microsome and granule membranes.