RESUMO
Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron-lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.
Assuntos
Epididimo/enzimologia , beta-Galactosidase/metabolismo , Animais , Líquidos Corporais/enzimologia , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Fluorometria , Coloide de Ouro , Masculino , Organelas/enzimologia , Organelas/ultraestrutura , Ratos , beta-Galactosidase/ultraestruturaRESUMO
In this study proteins were purified from rat sperm membranes which might be the high affinity sites for ligands of epididymal fluid other than the mannose-6-phosphate receptors. The sperm membrane proteins were solubilized and passed over an affinity column containing epididymal fluid proteins coupled to a matrix. Two bands in the range of 45-55 kDa were eluted from the column with fructose-6-phosphate but not with mannose-6-phosphate. Although the molecular weight of these proteins are similar to those of the cation-dependent phosphomannosyl receptors they are not related. These two proteins may correspond either to two different receptors or to forms of the same receptor that recognize ligands from rat epididymal fluid. Sequencing and identification of these proteins will be the aim of future studies.
Assuntos
Membrana Celular/química , Proteínas de Membrana/isolamento & purificação , Espermatozoides/ultraestrutura , Animais , Líquidos Corporais/química , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Epididimo/metabolismo , Frutosefosfatos/química , Immunoblotting , Masculino , Peso Molecular , Ratos , Receptor IGF Tipo 2/química , SolubilidadeRESUMO
This study demonstrates that alpha-mannosidase from rat epididymal fluid is a ligand for phosphomannosyl receptors on the sperm surface. This enzyme was bound to intact epididymal spermatozoa with high affinity and in saturable form, and the binding was inhibited by mannose-6-phosphate but not by phosphorylated derivatives of fructose. Treatment of the enzyme with sodium periodate inhibited the binding of alpha-mannosidase, confirming that a carbohydrate residue is involved in the interaction with spermatozoa. Evidence is also presented that the cation-independent phosphomannosyl receptors are responsible for the interaction with alpha-mannosidase. These findings suggest a new role for extracellular transport mediated by the mannose-6-phosphate receptor.
Assuntos
Manosidases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Espermatozoides/metabolismo , Animais , Líquidos Corporais/enzimologia , Epididimo/enzimologia , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Ratos , Receptor IGF Tipo 2 , alfa-ManosidaseRESUMO
The coat of clathrin-coated vesicles mostly consists of clathrin and adaptor complexes AP-1 or AP-2. Clathrin is released from the vesicles in an ATP-dependent fashion prior to their fusion with endosomes. In the present study we found that ATP strongly inhibits in vitro binding of cytosolic AP-2 to membranes of stripped vesicles, and promotes the release of endogenous AP-2 from clathrin-deprived coated vesicles. Both effects required hydrolysis of ATP. In contrast, binding of AP-1 to stripped vesicles was not affected by ATP, but was enhanced by GTP-gamma-S. These results point to an ATPase that promotes the release of AP-2 from clathrin-coated vesicles.
Assuntos
Acil Coenzima A/metabolismo , Adenosina Trifosfatases/metabolismo , Clatrina/metabolismo , Vesículas Revestidas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Membrana Celular/metabolismo , Citosol/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Hidrólise , Ligação Proteica/efeitos dos fármacosRESUMO
Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.
Assuntos
Glucuronidase/metabolismo , Espermatozoides/metabolismo , Fosfatase Alcalina/farmacologia , Sítios de Ligação , Humanos , Masculino , Manosefosfatos/farmacologia , Manosidases/farmacologia , Espermatozoides/efeitos dos fármacos , alfa-ManosidaseRESUMO
Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5% of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25% of total proteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.
Assuntos
Proteínas de Membrana/isolamento & purificação , Espermatozoides/química , Animais , Soluções Tampão , Membrana Celular/química , Membrana Celular/ultraestrutura , Detergentes , Eletroforese em Gel de Poliacrilamida , Masculino , Microscopia Eletrônica , Ratos , Espermatozoides/ultraestruturaRESUMO
The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.
Assuntos
Cálcio/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Calcimicina/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Quelantes/farmacologia , Clorpromazina/farmacologia , Antagonistas de Dopamina/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Galopamil/farmacologia , Imidazóis/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Morfogênese/efeitos dos fármacos , Triatoma , Trifluoperazina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismoRESUMO
Beta-galactosidase from rat epididymal fluid was purified by a combination of chromatographic techniques and precipitation with ammonium sulphate. Specific activity of the enzyme in the final precipitate was 18 times greater than in the original fluid, and it was practically free of N-acetyl-beta-D-glucosaminidase. A single major band was seen when the precipitate was analysed by sodium dodecylsulphate polyacrylamide gelectrophoresis (SDS-PAGE). The activity of the purified enzyme has an optimum at pH 4.5, and the temperature optimum is around 45 degrees C. The activity was inhibited by p-chloromercuribenzoic acid and ions such as Cd(II), Co(II), Cu(II) and Ag(I). Lactose does not appear to be a substrate for this enzyme.
Assuntos
Epididimo/enzimologia , beta-Galactosidase/isolamento & purificação , Animais , Precipitação Química , Cromatografia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Ratos , Temperatura , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/metabolismoRESUMO
Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65% of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9% of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.
Assuntos
Microtúbulos/química , Níquel/farmacologia , Tubulina (Proteína)/química , Animais , Química Encefálica , Centrifugação , Fenômenos Químicos , Físico-Química , Colchicina/metabolismo , Eletroforese em Gel de Poliacrilamida , Masculino , Ratos , Solubilidade , Tubulina (Proteína)/metabolismoRESUMO
Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65 of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9 of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.