RESUMO
A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.
Assuntos
Galactose/química , Sulfotransferases/química , Trissacarídeos/química , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Lactose/análogos & derivados , Lactose/química , Peso Molecular , Oligossacarídeos/química , Fosfoadenosina Fosfossulfato/química , Especificidade por Substrato , Sulfotransferases/sangue , Sulfotransferases/isolamento & purificação , SuínosRESUMO
Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.
Assuntos
Actinas/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/ultraestrutura , Animais , Galinhas , Cinética , Substâncias Macromoleculares , Microscopia Eletrônica , Modelos Estruturais , Músculos/metabolismo , Coelhos , Espectrometria de Fluorescência , Tropomiosina/isolamento & purificaçãoRESUMO
Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B-induced DNA fragmentation in a dose-dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.
Assuntos
Citocalasina B/toxicidade , Dano ao DNA , DNA/efeitos dos fármacos , Actinas/metabolismo , Animais , Proteínas de Transporte/toxicidade , Linhagem Celular , Permeabilidade da Membrana Celular , Núcleo Celular/efeitos dos fármacos , Citocalasina B/antagonistas & inibidores , Citocalasinas/metabolismo , Citocalasinas/toxicidade , DNA/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Humanos , Proteínas dos Microfilamentos/toxicidade , Peso Molecular , Ésteres de Forbol/farmacologia , Ratos , Fatores de TempoRESUMO
Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolymerization from the pointed end were measured in fluorescence assays using pyrenylactin filaments capped at the barbed end by villin. The initial pointed end depolymerization rate in the presence of tropomyosin averaged 56% of the control rate. Elongation from the pointed filament end in the presence of tropomyosin occurred at a lower free G-actin concentration, although the on rate constant, kappa p+, was not greatly affected. Furthermore, in the presence of tropomyosin, the free G-actin concentration was lower at steady state. Therefore, nonmuscle tropomyosin stabilizes the pointed filament end by lowering the off rate constant, kappa p-.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Tropomiosina/farmacologia , Animais , Biopolímeros , Galinhas , Mucosa Intestinal/análise , Cinética , Espectrometria de Fluorescência , Tropomiosina/metabolismoRESUMO
The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Glicoproteínas de Membrana , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/ultraestrutura , Tropomiosina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Sítios de Ligação , Ligação Competitiva , Galinhas , Mucosa Intestinal/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microvilosidades/metabolismoRESUMO
Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.
Assuntos
Actinas/metabolismo , Encéfalo/enzimologia , Intestinos/enzimologia , Isoenzimas/análise , Tropomiosina/análise , Aminoácidos/análise , Animais , Galinhas , Eletroforese em Gel de Poliacrilamida , Epitélio/enzimologia , Moela das Aves/enzimologia , Técnicas de Imunoadsorção , Cinética , Magnésio/farmacologia , Peso Molecular , Concentração Osmolar , ViscosidadeRESUMO
The enzyme GDP-D-mannose 4,6-dehydratase has been purified 1500-fold from porcine thyroid tissue. The enzyme exhibits a molecular mass of 251000 Da as determined by sedimentation techniques. Its subunit size was determined as 41500 Da by dodecyl sulfate gel electrophoresis. The enzyme has a Km of 3.3 microM with respect to GDP-D-mannose and appears specific with respect to this substrate. The enzyme appears to be inhibited by guanine nucleotides and by guanine nucleotide sugars. It is particularly susceptible to inhibition by GDP-L-fucose. It is suggested that this compound may have a physiological function as an end-product feedback inhibitor.
Assuntos
Hidroliases/isolamento & purificação , Glândula Tireoide/enzimologia , Animais , Eletroforese em Gel de Poliacrilamida , Hidroliases/antagonistas & inibidores , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato , SuínosRESUMO
The mechanism of GDP-D-mannose-4,6-dehydratase action with respect to loss of the C5 hydrogen has been established using GDP-D-[5-3H]-mannose as a substrate. This observation has been incorporated into a rapid assay for the enzyme based on the equilibration of 3H with the aqueous medium.
Assuntos
Hidroliases/metabolismo , Guanosina Difosfato Manose , Trítio , ÁguaRESUMO
The intestinal epithelial cell brush border (BB) is a useful model for nonmuscle cell motility. We studied regulation of BB motility by analyzing myosin phosphorylation and its association with the cytoskeleton. Our results demonstrate that myosin associates with the cytoskeleton only when it is dephosphorylated. Myosin light chain kinase substrates release myosin, phosphorylated and in the form of filaments, from the cytoskeleton. Although ITP and GTP serve as myosin ATPase substrates, they do not cause BB contraction, myosin release, or phosphorylation. Brush border contraction occurs with ATP or with a mixture of ITP and ATP gamma S. Therefore, phosphorylation regulates myosin association with the cytoskeleton, myosin is not bound at the actin-myosin binding site, and when phosphorylated, myosin forms filaments for movement.